slovenian society of human genetics

5th COLLOQUIUM OF GENETICS

Proceedings

PIRAN

September 23th, 2016

GENETIC SOCIETY SLOVENIAin collaboration with

SLOVENIAN SOCIETY OF HUMAN GENETICS

Colloquium of Genetics 2016

GENETIC SOCIETY OF SLOVENIA IN COLLABORATION WITH

THE SLOVENIAN SOCIETY OF HUMAN GENETICS

5th COLLOQUIUM OF GENETICS

Proceedings

Marine Biology Station Piran National Institute of Biology

Piran September 23rd 2016


2 | P a g e

Organizers

Genetic Society Slovenia in collaboration with The Slovenian Society of Human Genetics National Institute of Biology Marine Biology Station Piran University of Ljubljana Biotehnical Faculty

Faculty of Medicine University of Maribor

Faculty of Medicine Faculty of Chemistry and Chemical Technology

Editors Uroš Potočnik Katja Repnik Reviewers Branka Javornik Tanja Kunej Uroš Potočnik Maja Čemažar Jernej Jakše Damjan Glavač Design: Staša Jurgec Publisher: Genetic Society Slovenia, Ljubljana, September 2016 Number of issues: 60 USB keys

Contributing authors are responsible for proof‐reading corrections.


3 | P a g e

CIP - Kataložni zapis o publikaciji

Narodna in univerzitetna knjižnica, Ljubljana

575.111(082)(0.034.2)

616-056.7(082)(0.034.2)

COLLOQUIUM of Genetics (5 ; 2016 ; Piran)

Proceedings [Elektronski vir] / 5th Colloquium of Genetics, Piran, September

23rd 2016 ; [organizers] Genetic Society of Slovenia in collaboration with Slovenian

Society of Human Genetics ... [et al.] ; editors Uroš Potočnik, Katja Repnik. -

Ljubljana : Genetic Society of Slovenia, 2016

ISBN 978-961-93545-3-7

1. Potočnik, Uroš, 1969- 2. Slovensko genetsko društvo 3. Slovensko društvo za

humano genetiko

286511872


4 | P a g e

Members of boards

Scientific board Peter Dovč Damjan Glavač Branka Javornik Uroš Potočnik Jernej Jakše Darja Žgur Bertok Tanja Kunej Maja Čemažar

Organization board Katja Repnik Staša Jurgec Uroš Potočnik Andreja Ramšak Nataša Štajner


5 | P a g e

CONTENT

PROGRAM OF MEETING 7

LECTURES 8

OPENING LECTURE 9

Branka Javornik: STUDIES OF HOP-VERTICILLIUM PATHOSYSTEM 9

OPENING LECTURE 10

Marjanca Starčič Erjavec: GENETIC DIVERSITY OF Escherichia coli FROM GUT MICROBIOTA 10

ABSTRACTS AND PAPERS OF LECTURES 11

BIOTECHNOLOGY 11

Špela Kos: GENE ELECTROTRANSFER FOR DNA VACCINATION 12

Helena Volk: PRODUCTION OF RECOMBINANT EFFECTOR PROTEINS FROM Verticillium nonalfalfa 13

Mojca Juteršek: TOWARDS MOLECULAR GENETIC DELINIATION OF Synechocystis SPECIES 14

Ester Stajič: DEVELOPMENT OF CABBAGE HAPLOID INDUCER LINE WITH GENOME EDITING 20

Katja Guček: CRISPR/Cas9 GENOME EDITING IN RAPESEED 21

Sabina Belc: DISTRIBUTION ANALYSIS AND PRODUCTION OF FUNGAL ACTINOPORIN- AND PERFORIN-

LIKE PROTEINS 22

MOLECULAR BASIS OF DISEASES & GENOMICS 23

Miha Modic: TDP-43 SAFEGUARDS PLURIPOTENCY BY REGULATING ALTERNATIVE

POLYADENYLATION AND REPRESSING PARASPECKLES 24

Urša Lampreht Tratar: GENE ELECTROTRANSFER OF ANTIBIOTIC-FREE IL-12 PLASMID INDUCES

ANTITUMOR EFFECT IN B16F10 MELANOMAS 25

Katja Uršič: PERITUMORAL GENE ELECTROTRANSFER OF IL-12 AS ADJUVANT IMMUNOTHERAPY TO

INTRATUMORAL ELECTROCHEMOTHERAPY WITH CISPLATIN FOR TREATMENT OF MURINE B16F10

MELANOMA 26

Katarina Žnidar: THE EFFECT OF GENE ELECTROTRANSFER OF BLANK pDNA ON TUMOR CELLS'

SURVIVAL AND EXPRESSION OF CYTOSOLIC DNA SENSORS 27

Vasja Progar: FUNCTIONAL ENRICHMENT OF DIFFERENTIALLY EXPRESSED GENES IN HOP INFECTED

BY V. nonalfalfae 28

Kristina Marton: Verticillium nonalfalfae AND ITS CANDIDATE SECRETED EFFECTOR PROTEINS 29

Marija Rogar: POLYMORPHISMS IN SEGREGATION GENES INVOLVED IN THE DEVELOPMENT OF

GASTRIC CANCER IN SLOVENIAN POPULATION 30

Alja Zottel: GLIOBLASTOMA MULTIFORME AND NANOBODIES 31


6 | P a g e

POSTERS 32

ABSTRACTS AND PAPERS OF POSTERS 33

MOLECULAR BASIS OF DISEASES 33

Marko Vidak, Damjana Rozman, Mirjana Liović, Radovan Komel: SEARCH OF NEW GLIOBLASTOMA

STEM CELL MARKERS BY COMPARING MICROARRAYS DATA FROM THE NCBI GEO DATABASE, AND

EXPERIMENTAL VALIDATION OF THOSE MARKERS IN GLIOMA CELL LINES 34

Matej Rebek, Eva Jakljevič, Darja Žgur-Bertok, Marjanca Starčič Erjavec: PHYLOGENETIC GROUPS

AND clbAQ, usp, hlyA, cnf1 GENES AMONG COMMENSAL Escherichia coli STRAINS ISOLATED FROM

FECES OF HEALTHY HUMANS OF BOTH GENDERS AND BOTH AGE GROUPS 35

Tinkara Prijatelj, Danijela Vočanec, Tadej Pajič, Martina Fink, Irena Preložnik Zupan, Peter Černelč,

Radovan Komel, Tanja Kunej, Nataša Debeljak: NOVEL MOLECULAR-GENETIC DIAGNOSTIC TEST FOR

JAK2 NEGATIVE ERYTHROCYTOSIS 40

Rok Končnik, Barbara Krajnc, Jovan Krsteski, Uroš Potočnik: POLYMORPHISM rs1056837 IN CYP1B1

ENCODING AN ESTROGEN METABOLIZING ENZYME PRESENTS A RISK FOR ULM AMONG SLOVENIAN

WOMEN 41

Matjaž Deželak, Žan Hribar, Carina E. P. Kozmus, Uroš Potočnik: A POLYMORPHISM LOCATED IN

3’UTR OF PITHD1 NEAR CANNABINOID RECEPTOR 2 GENE CNR2 IS ASSOCIATED WITH SEVERE FORMS

OF CROHN’S DISEASE AND CNR1 GENE EXPRESSION 42

POPULATION GENETICS & GENOMICS 44

Taja Jeseničnik, Nataša Štajner, Sebastjan Radišek, Branka Javornik, Jernej Jakše: DISCOVERY OF

SMALL RNAs IN Verticilliun nonalfalfae 44

Matej Babič, Tanja Kunej, Borka Jerman-Blažič: NEW METHOD FOR STATISTICAL PATTERN

RECOGNITION USAGE IN CANCER ASSOCIATED microRNAs 45

Blaz Skrlj, Nina Pirih, Tanja Kunej: IDENTIFICATION OF NON-SYNONYMOUS POLYMORPHISMS

WITHIN REGIONS CORRESPONDING TO PROTEIN INTERACTION SITES 46

Marko Flajšman, Nataša Štajner, Igor Šantavec, Branka Javornik, Darja Kocjan Ačko: GENOTYPIZATION AND MORPHOLOGICAL CHARACTERIZATION OF SIX SLOVENIAN LANDRACES OF PROSO MILLET (Panicum miliaceum L.) 52

SPONSORS 53


7 | P a g e

PROGRAM OF MEETING

Registration 8.30 – 9.00

Opening of the 5th COLLOQUIUM OF GENETICS

9.00 – 10.00

OPENING LECTURE:

Chairman: Uroš Potočnik

Branka Javornik: Studies of hop-Verticillium pathosystem

Marjanca Starčič Erjavec: Genetic diversity of Escherichia coli from gut microbiota

9.10 – 10.00

9.10 – 9.35 9.35 – 10.00

Biotechnology

Chairmen: Sabina Berne in Jernej Jakše

10.00 – 11.30

Špela Kos: Gene electrotransfer for DNA vaccination 10.00 – 10.15

Helena Volk: Production of recombinant effector proteins from Verticillium nonalfalfae 10.15 – 10.30

Mojca Juteršek: Towards molecular genetics deliniation of Synechocystis species 10.30 – 10.45

Ester Stajič: Development of cabbage haploid inducer line with genome editing 10.45 – 11.00

Katja Guček: CRISPR/Cas9 genome editing in rapeseed 11.00 – 11.15

Sabina Belc: Distribution analysis and production of fungal actinoporin- and perforin-like proteins

11.15 – 11.30

Coffee break and poster viewing 11.30 – 11.50

Molecular Basis of Diseases and Genomics

Chairmen: Tanja Kunej in Marjanca Starčič Erjavec

11.50 – 13.50

Miha Modic: TDP-43 safeguards pluripotency by regulating alternative polyadenylation

and repressing paraspeckles

11.50 – 12.05

Urša Lampreht Tratar: Gene electrotransfer of antibiotic-free IL-12 plasmid induces

antitumor effect in B16F10 melanomas

12.05 – 12.20

Katja Uršič: Peritumoral gene electrotransfer of IL-12 as adjuvant immunotherapy to

intratumoral electrochemotherapy with cisplatin for treatment of murine B16F10

melanoma

12.20 – 12.35

Katarina Žnidar: The effect of gene electrotransfer of blank pDNA on tumor cells' survival

and expression of cytosolic DNA sensors

12.35 – 12.50

Vasja Progar: Functional enrichment of differentially expressed genes in hop infected by

V. nonalfalfae

12.50 – 13.05

Kristina Marton: Verticillium nonalfalfae and its candidate secreted effector proteins 13.05 – 13.20

Marija Rogar: Polymorphisms in segregation genes involved in the development of gastric cancer in Slovenian population

13.20 – 13.35

Alja Zottel: Glioblastoma multiforme and nanobodies 13.35 – 13.50

Lunch (self-service bar), poster viewing 13.50 – 15.00

Meeting of the Genetic Society Slovenia with announcement of the best lecture and the best poster awards

15.00 – 16.00


8 | P a g e

LECTURES


9 | P a g e

OPENING LECTURE

STUDIES OF HOP-VERTICILLIUM PATHOSYSTEM Branka Javornik

University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

ABSTRACT

The studies address Verticillium resistance in plants, working on the hop-Verticillium nonalfalfea (Vna)

pathosystem. This work includes two broad aims.

First, to improve hop resistance to Verticillium wilt. This work includes mapping of gene(s) or QTLs for

Verticillium resistance and the development of molecular markers linked to resistance for application

in hop breeding.

The second aim is to improve the management of wilt disease based on an understanding of the

mechanisms of host- pathogen interaction and the pathogenicity of Vna. This work has focused on

studies of the hop-Vna interaction on proteome and transcriptome levels to search for hop factors

implicated in resistance and Vna factors related to virulence. On the pathogen side, Vna has been being

further studied by comparison of proteomes and whole genome sequences of Vna isolates with

different virulence, and functional analysis of predicted candidate genes.


10 | P a g e

OPENING LECTURE

GENETIC DIVERSITY OF Escherichia coli FROM GUT MICROBIOTA

Marjanca Starčič Erjavec

University of Ljubljana, Biotechnical Faculty, Department of Biology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

ABSTRACT

Mammals have a complex gut microbiota shaped by intestinal anatomy, function and diet1.

Escherichia coli (E. coli) is one of the first colonizers of the mammalian gut and afterwards part of the

normal gut microbiota. It usually coexists with its host in mutual benefit. Nevertheless, it is known

that the intestinal microbiota can also be a reservoir of extraintestinal pathogenic variants of E. coli

(ExPEC) that can cause disease infections at different extraintestinal anatomic sites (e. g. urinary

tract, skin and soft-tissue infections)2. Further, it is known that animals (e. g. cats, dogs and birds) can

be a potential reservoir of ExPEC3. In last years our studies have been focused on the virulence

potential for ExPEC among E. coli from gut microbiota from different hosts. The lecture will present

an overview of the published4, 5 and recently obtained data.

REFERENCES 1. Stevens CE, Hume ID. The mammalian gastrointestinal tract. In: Comparative physiology of the vertebrate digestive

system. 2nd ed. Cambridge University Press, Cambridge, 2004, 46-92.

2. Leimbach A, Hacker J, Dobrindt U. E. coli as an all-rounder: the thin line between commensalism and pathogenicity. Curr.

Top. Microbiol. Immunol. 2013, 358, 3-32.

3.Bélanger L, Garenaux A, Harel J, Boulianne M, Nadeau E, Dozois CM. Escherichia coli from animal reservoirs as a potential

source of human extraintestinal pathogenic E. coli. FEMS Immunol. Med. Microbiol. 2011, 62, 1-10.

4. Starčič Erjavec M, Žgur-Bertok D. Virulence potential for extraintestinal infections among commensal Escherichia coli

isolated from healthy humans – the Trojan horse whitin our gut. FEMS Microbiol. Lett. 2015, 362, 1-9.

5. Vadnov M, Barbič D., Žgur-Bertok D, Starčič Erjavec M. Escherichia coli isolated from feces of brown bears (Ursus arctos)

have a lower prevalence of human extraintestinal pathogenic E. coli virulence-associated genes. Can. J. Vet. Res. 2016, In

press.


11 | P a g e

ABSTRACTS and PAPERS of LECTURES

BIOTECHNOLOGY

Špela Kos

Gene electrotransfer for DNA vaccination

Helena Volk

Production of recombinant effector proteins from Verticillium nonalfalfae

Mojca Juteršek

Towards molecular genetics deliniation of Synechocystis species

Ester Stajič

Development of cabbage haploid inducer line with genome editing

Katja Guček

CRISPR/Cas9 genome editing in rapeseed

Sabina Belc

Distribution analysis and production of fungal actinoporin- and perforin-like proteins


12 | P a g e

Corresponding author: Špela Kos, [email protected]

Abstract

GENE ELECTROTRANSFER FOR DNA VACCINATION

Spela Kos1, Kevin Vanvarenberg2, Tanja Dolinsek1, Maja Cemazar1, 3, Jure Jelenc4, Véronique Préat2, Gregor Sersa1, Gaëlle Vandermeulen2

1Institute of Oncology Ljubljana, Department of Experimental Oncology, Slovenia 2Université Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Avenue E.,

Belgium 3University of Primorska, Faculty of Health Sciences, Slovenia 4Iskra Medical d.d.o., Slovenia

ABSTRACT

A number of notable technology advances in DNA vaccination over the past few years have led to the

resurgence of this field as a promising treatment modality. Among these advancements are improved

physical methods of naked DNA delivery to cells. Of these, gene electrotransfer is considered as a safe

and efficient method to deliver the plasmids to target cells or tissues1. Among the potential targets,

skin is an easy accessible and immunocompetent tissue, which makes it an attractive target for gene

electrotransfer2. Our study focused on gene electrotransfer of model DNA vaccine coding for the

ovalbumin (OVA) protein, delivered into the mouse skin. For this purposes the non-invasive multi-

electrode array (MEA) was used3. The efficiency of the gene expression and the activation of immune

response against delivered antigen were followed. The results demonstrated strong gene expression

and an efficient delivery of DNA vaccine. The use of MEA to deliver the ovalbumin plasmid generated

a strong immune response, as evidenced by the presence of antibodies in the serum, the IFN-gamma

response and the delayed tumor growth when the mice were challenged with B16-OVA cells. The

described method of gene electrotransfer by MEA electrode to skin proved as a promising approach

to deliver the plasmids coding for the therapeutic molecules. It sets the stage for the further

development of electroporation mediated delivery of DNA vaccines against infectious agents,

autoimmunity or for cancer therapy.

REFERENCES 1. Lambricht L, Lopes A, Kos S, Sersa G, Preat V, Vandermeulen G. Clinical potential of electroporation for gene therapy and

DNA vaccine delivery. Expert Opinion Drug Deliv. 2015, 13, 295-310.

2. Gothelf A, Gehl J. Gene electrotransfer to skin; review of existing literature and clinical perspectives. Curr Gene Ther.

2010, 10, 287-299.

3. Kos S, Blagus T, Cemazar M, Jelenc J, Sersa G. Utilization of multi-array electrodes for delivery of drugs and genes in the

mouse skin. IFMBE proceedings. 2015, 53, 321-324.


13 | P a g e

Corresponding author: Helena Volk, [email protected]

Abstract

PRODUCTION OF RECOMBINANT EFFECTOR PROTEINS FROM Verticillium nonalfalfae

Helena Volk 1, Marko Dolinar 2, Branka Javornik 1, Sabina Berne 1

1Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Slovenia 2Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia

ABSTRACT

Verticillium nonalfalfae (Vna) is a phytopathogenic fungus causing Verticillium wilt on various plants,

including hop (Humulus lupulus L.). The fungus enters the plant through the roots and continues to

spread into the stem of the plant. To colonise the host plant successfully, Vna secrets a repertoire of

effector proteins that protect the fungus from the plant immune system or modulate the host plant

physiology.

Unravelling how effectors function in the host organism leads to better understanding of the plant-

pathogen interactions and may facilitate the discovery of host plant proteins involved in resistance to

the pathogen. Recombinant proteins expressed in Escherichia coli and Pichia pastoris have been used

for biochemical characterisation and functional analysis of effectors.

The current study focuses on two effector proteins, VnaSSP4.2 and VnaCBP8.213, that are highly

expressed during infection of hop. The two effectors without signal peptide were cloned to pET32a

expression vector and produced in E. coli. Various parameters were tested to obtain a high amount of

soluble recombinant proteins: bacterial strains Shuffle (optimized for the formation and maintenance

of disulphide bonds) and BL21(DE3)pLysS (drives protein production in a reducing environment),

optimal expression temperature and concentration of IPTG inducer. Protein expression was examined

by Western blot analysis, and the optimal parameters for purification of recombinant protein using Ni-

NTA affinity chromatography on FPLC system were chosen.

Our results confirm that the parameters for efficacious recombinant protein production are protein to

protein dependent, with bacterial strain and temperature during expression being the most significant

factors.


14 | P a g e

Corresponding author: Marko Dolinar, [email protected]

Article

TOWARDS MOLECULAR GENETIC DELINIATION OF Synechocystis SPECIES

Mojca Juteršek, Marina Klemenčič, Marko Dolinar

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia

Identification and classification of cyanobacteria have been traditionally based on their morphological features, which

often lead to misidentifications and false classifications. To overcome variable morphological criteria, DNA sequencing is

becoming the most widely applied molecular method in the identification and cataloguing of cyanobacteria. We focused

on the unicellular genus Synechocystis that includes the model cyanobacterium Synechocystis sp. PCC 6803, broadly used

in biotechnology and synthetic biology. Little is known about the distribution of this strain and of its relatives in the

environment, which might be important for biosafety assessments in biotechnology and synthetic biology. Two genomic

regions, the variable part of the 16S rRNA coding region and the 16S – 23S internal transcribed spacer (ITS), were

amplified by PCR from 11 Synechocystis members, cloned and sequenced. Comparison of our sequencing data with

previously published sequences indicates that the current Synechocystis genus is highly heterogeneous, but that the ITS

region alone reflects the phylogenetic positioning of strains very well.

INTRODUCTION

Cyanobacteria represent an evolutionary and ecologically important group of photosynthetic

microorganisms. Furthermore they are also becoming increasingly studied for their use in

biotechnology, e. g. for production of biofuels. This creates the need for use of recombination

technology or synthetic biology, resulting in GMOs that pose potential biosafety hazards that need to

be identified and minimized. One of such hazards is the possibility of horizontal gene transfer and

homologous recombination, two phenomena proven to be significant among prokaryotes1. Since the

most broadly used cyanobacterial strain in biotechnology and synthetic biology is Synechocystis sp.

PCC 6803 which is also naturally competent for genetic transformation2, we wished to know whether

there are any close relatives of the strain present in aquatic environments and planned to develop a

DNA barcoding approach specifically for these unicellular cyanobacteria.

Most widely used DNA-based method for identification of cyanobacterial species is 16S rRNA

gene analysis, using the assumption that individuals of the same species share greater sequence

similarity than individuals of different species3. Although overall evolution of 16S rRNA gene is rather

slow, there are regions that are more variable and enable discrimination of distantly as well as

closely related organisms. With its variable length and number, rRNA ITS region has become a

popular tool in identification and classification of cyanobacteria4. Our goal was therefore to

investigate possibilities for eventual ITS-based molecular tool for discrimination between species and

strains of the Synechocystis genus.

METHODS

Eleven Synechocystis strains were obtained from different culture collections (except for

Synechocystis nigrescens that was obtained from a supplier of teaching consumables), as listed in

Table 1.

Table 1: List of analysed strains. SAG - Sammlung von Algenkulturen Göttingen, CCAP -

Culture Collection of Algae and Protozoa, CCALA - Culture Collection of Autotrophic Organisms, PCC -

Pasteur Culture Collection, Carolina = Carolina Biological Supply Company.


15 | P a g e

Species Strain label

Synechocystis aquatilis SAG 90.79

Synechocystis bourrellyi CCAP 1480/1

Synechocystis fuscopigmentosa CCALA 810

Synechocystis limnetica CCAP 1480/5

Synechocystis minuscula SAG 258.80

Synechocystis nigrescens Carolina

Synechocystis pevalekii SAG 90.79

Synechocystis salina CCALA 192

Synechocystis sp. CCAP 1480/4

Synechocystis sp. PCC 6714

Synechocystis sp. PCC 6803

Cells from 1 ml of culture were pelleted at 5000 g for 5 min and boiled for 10 min at 95°C.

Lysates were used for PCR using Taq polymerase. For amplification of the variable region of 16S

rDNA, primers CYA106F (5'-CGGACGGGTGAGTAACGGTGA-3'),

CYA781Ra (5'-GACTACTGGGGTATCTAAATCTTATT-3') and

CYA781Rb (5'-GACTACAGGGGTATCTAATCCCTTT-3') were used. For amplification of ITS regions we

used primers CSIF (5'-GTCACGCCCGAAGTCGTTAC-3') and ULR (5'- CCTCTGTGTGCCTAGGTATC-3').

Reverse primers for 16S rDNA amplification (CYA781Ra in CYA781Rb) were used in equimolar

quantities. All used primers are marked on ribosomal RNA operon scheme in Figure 1.

Figure 1: Scheme of partial rrn operon with marked primer positions. Nucleotide positions

are labelled for reference as deduced from Synechocystis sp. PCC 6803 genome.

PCR products were resolved on 1.2% or 1.5% agarose gels and visualized using ethidium

bromide. After electrophoresis, PCR products were excised from agarose gels and purified using

GeneJet Gel Extraction Kit (Thermo Scientific). Purified products were ligated into pJET1.2 using

CloneJET PCR Cloning Kit (Thermo Scientific). After transformation of competent Escherichia coli

DH5α cells and plating onto selective media, plasmid DNA was isolated from overnight cultures of

one to several independent clones using Plasmid MiniPrep Kit (Thermo Scientific). Sequencing was

performed by Macrogen Europe.

All the sequences were compared to the non-redundant dataset of the GenBank collection

using BLASTN. For other sequence analyses EMBOSS Water and Clustal Omega were used, both at

the EMBL-EBI web server. For multiple alignments of 16S sequences we utilized RDP Aligner. We built


16 | P a g e

neighbourhood-joining tree using MEGA version 6 applying the Jukes-Cantor model. Bootstrap

resampling using 500 replicates was performed to test the robustness of the trees.

RESULTS

Agarose gel electrophoresis of ITS amplicons showed great diversity of lengths as can be seen

from Figure 2. According to ITS lengths, Synechocystis members can be roughly divided into four

groups. The shortest regions were found in S. aquatilis and S. fuscopigmentosa (group A). Most of the

analysed representatives belong to the group with lengths similar to PCC 6803 and PCC 6714 (S.

minuscula, S. salina, S. sp. CCAP 1480/4). Group C with an intermediate size ITS region was

represented by S. pevalekii and group D with very long ITS regions by S. limnetica and S. bourrellyi.

These differences allow for a rapid PCR-based discrimination between some of the Synechocystis

members without sequencing.

Figure 1: PCR amplification products of ITS regions for the 10 Synechocystis strains using CSIF

and ULR primers, resolved on 1% agarose gel. (1) Synechocystis sp. PCC 6803, (2) Synechocystis sp.

PCC 6714, (3) Synechocystis sp. CCAP 1480/4, (4) Synechocystis salina CCALA 192, (5) Synechocystis

minuscula SAG 258.80, (6) Synechocystis aquatilis SAG 90.79, (7) Synechocystis fuscopigmentosa

CCALA 810, (8) Synechocystis pevalekii SAG 91.79, (9) Synechocystis limnetica CCAP 1480/5, (10)

Synechocystis bourrellyi CCAP 1480/1. ITS region of Synechocystis nigrescens could not be amplified

using CSIF and ULR primers.

Since the observed ITS length heterogeneity exceeded expected differences among species

of the same genus, we performed sequencing and BLAST searches. We found that Synechocystis

members whose ITS lengths differed from the length obtained with PCC 6803, showed higher

relatedness to members of different genera. Group B seemed to be related to Geminocystis and

Cyanobacterium genera, group C to Chamaesiphon and group D to Synechococcus. Results of BLAST

search with highest scoring strains and their identity to our sequences are presented in Table 2.

To confirm results obtained with ITS regions, we amplified, sequenced and performed a

BLAST search of 16S rRNA gene variable regions and found results supportive of previous findings.

With concatenated 16S and ITS sequences we prepared a phylogenetic tree that shows clustering

into 4 groups as can be seen in Figure 3.


17 | P a g e

Figure 2: A phylogenetic tree for the analysed members of the Synechocystis genus. Tree is

based on our sequencing results of the 16S rRNA and ITS regions combined (S. nigrescens is not

included as its ITS region could not be amplified). For comparison, sequence data for PCC 6714 and

PCC 7509 as deposited in GenBank were included.


18 | P a g e

Table 1: Results of BLAST search with highest scoring strains (GenBank ID numbers of each strain are in brackets) and their identity to our 16S rRNA

and ITS sequences. When the highest score was that of the same strain and collection, the second highest score is listed. Results for fully sequenced S. sp.

PCC 6803 and S. sp. PCC 6714 strains are also not shown, since they showed sequences from genomes of both strains as highest scores, as expected.

Different results for 16S and ITS for one strain are due to differences in deposited sequences, since for some strains only either 16S or ITS sequence is

available. * S. salina shows highest identity to Gloecapsa alpicola FACHB-400, which has been recently reclassified twice, firstly to Synechocystis and then to

Geminocystis genus.

Species/Strain

Highest BLAST scores

16S ITS

Strain and GenBank ID Identity Strain and GenBank ID Identity

S. aquatilis Cyanobacterium aponinum KSU-WH-5 (KT807478.1),

lklSCC30 (KM438201.1) and PCC 10605 (CP003947.1)

97.0% Cyanobacterium aponinum PCC 10605 (CP003947.1) 95.0%

S. bourrellyi Synechococcus elongatus CCAP 1479/1B (KM020008.1),

Synechococcus sp. CCAP 1479/10 (HE975006.1), PCC 7009

(AM709628.1), EW15 (DQ275602.1) and BO8806

(AF317072.1)

99.6% Synechococcus sp. PCC 7009 (AM709628.1) 99.8%

S. fuscopigmentosa Geminocystis sp. NIES-3709 (AP014821.1) 98.9% Geminocystis sp. NIES-3709 (AP014821.1) 96.8%

S. limnetica Synechococcus sp. MA0607K (FJ763779.1) 99.2% Prochlorococcus marinus MIT9313 (BX548175.1) 87.6%

S. minuscula Synechocystis salina LEGE 06155 (HQ832911.1) and

Synechocystis cf. salina LEGE 07073 (HM217083.1)

97.4% Gloeothece sp. PCC 6909 (CCAP 1480/4,

(HE975009.1)

80.0%

S. nigrescens Synechocystis sp. SAG 37.92 (KM020010.1) 99.8% / /

S. pevalekii Chamaesiphon subglobosus PCC 7430 (AY170472.1) 99.6% Chamaesiphon minutus PCC 6605 (CP003600.1) 90.0%

S. salina* Gloeocapsa alpicola FACH-400 (JX872524.1) and

Gloeothece sp. PCC 6909 (HE975009.1)

99.6% Gloeothece sp. PCC 6909 (HE975009.1) 98.8%

S. sp CCAP 1480/4 Gloeocapsa alpicola FACHB-400 (JX872524.1) 99.7% Synechocystis sp. PAK12 (EF555570.1) 93.2%


19 | P a g e

DISCUSSION

Our original goal was to develop DNA barcoding for Synechocystis genus. The present analysis

showed great genetic heterogeneity the of current Synechocystis genus especially in their ITS regions,

which is a good basis for development of discrimination methods, either by agarose gel

electrophoresis or with use of ITS-specific primers or for developing sequence-based approach.

Also, our research greatly expanded the range of Synechocystis members with available

sequences which could eventually contribute to a more precise taxonomic delineation of the genus.

Namely, variability in 16S rRNA and ITS genomic regions indicates that some current Synechocystis

members are too genetically different to be assigned to this genus. Taxonomic inconsistencies are

not new in Cyanobacteria phylum since cyanobacterial systematics was traditionally based on

morphology alone. This turned out to be problematic because of cryptic variability, lack of

morphological differences and convergent evolution5. Also, cells can change morphology in varying

growth conditions or when transferred from natural to laboratory growth conditions6,7. Mislabelling

of strains in culture collections therefore cannot be excluded as it has also been noted before that

more than a half of the strains in culture collections are probably incorrectly identified8.

Since the introduction of genetic methods to the field of cyanobacterial taxonomy, many

systematic reconsiderations were needed, but no extensive research has been done for

Synechocystis genus so far. Our current results represent a solid basis for taxonomic reconsideration

of Synechocystis and related cyanobacterial genera.

REFERENCES 1. Achtman, M. and Wagner, M. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 2008,

6, 431-440.

2. Yu, Y., You, L., Liu, D., Hollinshead, W., Tang, Y. J. and Zhang, F. Development of Synechocystis sp. PCC 6803 as a

phototrophic cell factory. Marine Drugs 2013, 11, 2894-2916.

3. Eckert, E. M., Coates, R. C., Coci, M. and Callieri, C. Does a barcoding gap exist in prokayotes? Evidences from species

delimitation in cyanobacteria. Life 2015, 5, 50-64.

4. Smit, S., Widmann, J. and Knight, R. Evolutionary rates vary among rRNA structural elements. Nucl. Acids Res. 2007, 35,

3339-3354.

5. Dvořák, P., Poulíčková, A., Hašler, P., Belli, M., Casamatta, D. A. and Papini, A. Species concepts and speciation factors in

cyanobacteria, with connection to the problems of diversity and classification. Biodiver. Conserv. 2015, 24, 739-757.

6. Castenholz, R. W. and Waterbury, J. B. Oxygenic phototrophic bacteria. Group I. Cyanobacteria. In: Staley, J. T., Bryant, M.

P., Pfenning, N. and Holt, J. G (eds) Bergey's manual of systematic bacteriology. 1989. Baltimore: Williams and Wilkins,

1710-1789.

7. Moro, I., Rascio, N., La Rocca, N., Di Bella, M and Andreoli C. Cyanobacterium aponinum, a new cyanoprokaryote from the

microbial mat of Euganean thermal springs (Padua, Italy). Algol. Studies 2007, 123, 1-15.

8. Komárek, J. and Anagnostidis, K. Modern approach to the classification system of Cyanophytes 4 – Nostocales. Algol.

Studies 1989, 56, 247-345.


20 | P a g e

Corresponding author: Ester Stajič, [email protected]

Abstract

DEVELOPMENT OF CABBAGE HAPLOID INDUCER LINE WITH GENOME EDITING

Ester Stajič1, Jana Murovec1, Borut Bohanec1

1 University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Slovenia

ABSTRACT

For plant breeding, induction of haploids is one of the fastest techniques for the production of hybrids

because homozygous lines can be made in one generation. One of techniques for production of

parental homozygous plants is pollination with inducer lines which can be produced with different

point mutations in the centromere-specific histone H3 variant (CENH3)1. Because CENH3 is highly

conserved, the same point mutations could be generated by genome editing also in cabbage.

Therefore, our aim was to develop a protocol for genome editing of CENH3 gene in cabbage using

CRISPR/Cas9 system. For that purpose, three sgRNAs targeting CENH3 in cabbage were designed and

expression vectors were constructed that would be introduced in cabbage cells through two different

transformation techniques.

We developed a protocol for Agrobacterium tumefaciens mediated transformation and tested

different transformation parameters, like the type of explant (hypocotyl, leaf, petiole), concentration

of selective antibiotic and duration of selection (1- 4 weeks).

For protoplast transformation, a protocol for protoplast isolation and regeneration was first developed

using immobilization in calcium alginate layers2. We tested the effect of incubation time and source of

protoplasts on protoplasts isolation efficiency. For optimization of protoplasts regeneration protocol,

protoplasts were cultured at different densities and after formation of calli, they were transferred to

medium supplemented with or without different growth regulators.

In the future, we plan to use the developed techniques to first edit cabbage CENH3 gene and later to

use them for editing of other targets.

REFERENCES 1. Karimi-Ashtiyani R, Ishii T et al. Point mutation impairs centromeric CENH3 loading and induces haploid plants. PNAS.

2015, 112, 36, 11211-11216. 2. Kielkowska A, Adamus A. An alginate-layer technique for culture of Brassica oleracea L. protoplasts. In vitro Cell.Dev.Biol.-

Plant. 2012, 48, 265-273.


21 | P a g e

Corresponding author: Katja Guček, [email protected]

Abstract

CRISPR/Cas9 genome editing in rapeseed

Katja Guček1, Sabina Mazinjanin1, Jana Murovec1

1University of Ljubljana, Biotechnical Faculty, Agronomy Department, Slovenia

ABSTRACT

Rapeseed (Brassica napus L.) is one of the most important crops for oil production and a model

organism for microspore embryogenesis studies. The aim of our work was to develop delivery methods

for ribonucleoprotein-guided endonucleases (RNPs) into rapeseed cells which could be later used for

genome editing without integrating foreign genetic material1 into the rapeseed genome. For this

purpose, two different cell types (microspores and protoplasts) and two different delivery methods

were tested.

First, isolation protocols for highly viable protoplasts and highly embryogenic microspores were

optimized. Up to 84% viable protoplasts were produced with overnight incubations of etiolated

hypocotyls and the most embryogenic microspores (at late uninucleate to early binucleate

developmental stage) were obtained from 3-4 mm flower buds. Several sgRNAs were constructed and

in vitro synthesised targeting exons of phytoene desaturase gene (pds) encoding an important enzyme

in the carotenoid biosynthetic pathway which mutation results in albino and dwarf phenotypes4.

Before transformation, sgRNA and Cas9 were preassembled in molar ratios from 1:10 to 1:205. They

were introduced in microspores with electroporation and in protoplasts with PEG. After 24 hours, on

target mutations were detected using T7E1 endonuclease assay, restriction enzymes site loss assay

and Sanger sequencing. Preliminary results showed targeted mutagenesis of protoplasts in the region

of exon 1 of the pds gene using the T7E1 assay. New experiments are currently underway. With further

optimization, these techniques could be used for basic research and for plant breeding.

REFERENCES 1. Kanchiswamy CD. DNA-free genome editing methods for targeted crop improvement, Plant Cell Rep.2016, 35,

1469–1474

2. Custers J. “Microspore culture in rapeseed (Brassica napus L.),” in Doubled Haploid Production in Crop Plants, eds

Maluszynski M, Kasha KJ, Forster BP, Szarejko I. Dordrecht,Kluwer Academic Publishers 2003, 185–193.

3. Wang YP, Sonntag K, Rudlogg E. Development of rapeseed with high erucic acid content by asymmetric somatic

hybridization between Brassica napus and Crambe abyssinica. Theoretical and Applied Genetics 2003, 106, 1147-

1155.

4. Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ. Disruption of phytoene desaturase gene results in albino and

dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res. 2007,

17, 471-82.

5. Woo JW, Kim J, Kwon SI, Corvalan C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS. DNA-free genome editing in

plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol.2015 33(11):1162–1164.


22 | P a g e

Corresponding author: Sabina Belc, [email protected]

Abstract

DISTRIBUTION ANALYSIS AND PRODUCTION OF FUNGAL ACTINOPORIN- AND PERFORIN-

LIKE PROTEINS

Sabina Belc1,2, Nada Kraševec1, Gregor Anderluh1

1National Institute of Chemistry, Laboratory for Molecular Biology and Nanobiotechnology, Slovenia 2University of Ljubljana, Biotechnical Faculty, Biotechnology, Slovenia

ABSTRACT

Pore-forming proteins are currently of great interest due to their role in fungal pathogenicity, which is

recognized as a serious threat to food safety1 and public health2. The aim of our study was to analyze

the prevalence of aegerolysins, MACPF (Membrane Attack Complex component/PerForin) and NLP

(NEP-1-like protein) proteins within the fungi kingdom. We attempted to express proteins B473 (a PlyB

homolog) and NLP from fungus Aspergillus niger in bacterium Escherichia coli, in order to confirm the

results of bioinformatic analysis, and characterize the properties of the proteins.

We found that the prevalence of the proteins does not correlate with the fungal lifestyle. Individual

organisms possess several different copies of the genes encoding the proteins under study, which are

spread throughout the genome and the phylogenetic tree. This suggests the possibility of different

gene donors. Synteny analysis showed that in 38 cases, homolog PlyA is found adjacent to homolog

PlyB, like in the fungus Pleurotus ostreatus.

There is an increasing body of evidence to suggest that HGT is an important mechanism in eukaryotic

genome evolution3. Researchers connected HGT to emergence of new diseases2 and expansion of host

range4. We used in silico methods to discover a potential horizontal gene transfer event in fungus A.

niger, and noted repeated sequences in its vicinity that could be due to transposon activity.

This study revealed some interesting starting points for further research. It would be advantageous to

optimize the production and isolation of target pore-forming proteins, as they offer a great potential

for biotechnological innovations5,6.

REFERENCES 1. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ. Emerging fungal threats to animal, plant

and ecosystem health. Nature. 2012, 484, 7393: 186–194.

2. Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida

albicans and Aspergillus fumigatus. Journal of clinical microbiology. 2004, 42, 10: 4419–4431.

3. Fitzpatrick DA. Horizontal gene transfer in fungi. FEMS Microbiology Letters. 2012, 329, 1: 1–8.

4. Mehrabi R, Bahkali AH, Abd-Elsalam KA, Moslem M, Ben M’barek S, Gohari AM, Jashni MK, Stergiopoulos I, Kema GHJ,

de Wit PJGM. Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range. FEMS

Microbiology Reviews. 2011, 35, 3: 542–554.

5. Majd S, Yusko EC, Billeh YN, Macrae MX, Yang J, Mayer M. Applications of biological pores in nanomedicine, sensing,

and nanoelectronics. Current Opinion in Biotechnology. 2010, 21, 4: 439–476

6. Provoda CJ, Lee KD. Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules.

Advanced Drug Delivery Reviews. 2010, 41, 2: 209–221.


23 | P a g e

ABSTRACTS of LECTURES

MOLECULAR BASIS OF DISEASES & GENOMICS

Miha Modic

TDP-43 safeguards pluripotency by regulating alternative polyadenylation and

repressing paraspeckles

Urša Lampreht Tratar

Gene electrotransfer of antibiotic-free IL-12 plasmid induces antitumor effect in

B16F10 melanomas

Katja Uršič

Peritumoral gene electrotransfer of IL-12 as adjuvant immunotherapy to intratumoral

electrochemotherapy with cisplatin for treatment of murine B16F10 melanoma

Katarina Žnidar

The effect of gene electrotransfer of blank pDNA on tumor cells' survival and

expression of cytosolic DNA sensors

Vasja Progar

Functional enrichment of differentially expressed genes in hop infected by V.

nonalfalfae

Kristina Marton

Verticillium nonalfalfae and its candidate secreted effector proteins

Marija Rogar

Polymorphisms in segregation genes involved in the development of gastric cancer in

Slovenian population

Alja Zottel

Glioblastoma multiforme and nanobodies


24 | P a g e

Corresponding author: Miha Modic, [email protected]

Abstract

TDP-43 SAFEGUARDS PLURIPOTENCY BY REGULATING ALTERNATIVE POLYADENYLATION

AND REPRESSING PARASPECKLES

Miha Modic1,2, Gregor Rot3, Markus Grosch1, Tjasa Lepko1, Dmitry Shaposhnikov1, Ejona Rusha1, Davide Cacchiarelli4, Boris Rogelj5,6, Christian von Mering3, Alexander Meissner4, Jernej Ule7,8*, Micha Drukker1* 1Institute of Stem Cell Research, Helmholtz Zentrum München, Munich, Germany 2International Max Planck Research School, Max Planck Institute of Biochemistry, Munich, Germany 3Institute of Molecular Life Sciences and Swiss Bioinformatic Institute, Zurich Switzerland 4Broad Institute of Harvard University/MIT, Boston, USA 5Department of Biotechnology, Jožef Stefan Institute, and Biomedical Research Institute BRIS, Ljubljana, Slovenia 6Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia 7Institute of Neurology, Department for Molecular Neuroscience, University College London, United Kingdom 8Francis Crick Institute, London, United Kingdom

ABSTRACT

A fundamental question of stem cell biology is what dissolves the self-renewal program to facilitate

exit from pluripotency. Here we show that TDP-43 regulates alternative polyadenylation (APA) during

early differentiation, including of the pluripotency factor SOX2. A decrease of TDP-43 during early

differentiation leads to loss of SOX2 due to the coupled action of miR-21. Hence, overexpression or

reduction of TDP-43 promotes somatic cell reprogramming/pluripotency or differentiation,

respectively. Moreover, TDP-43 stimulates production of an unstable short isoform of the lncRNA

NEAT1. Reduced TDP-43 allows formation of the stable full-length isoform of NEAT1, which is required

for efficient differentiation by scaffolding the nuclear domains named paraspeckles. Taken together,

our work reveals a post-transcriptional axis that stabilizes the states of pluripotency and differentiation

independently of any specific lineage.


25 | P a g e

Corresponding author: Ursa Lampreht Tratar, ulamprehtonko-i.si

Abstract

GENE ELECTROTRANSFER OF ANTIBIOTIC-FREE IL-12 PLASMID INDUCES ANTITUMOR

EFFECT IN B16F10 MELANOMAS

Ursa Lampreht Tratar1, Luisa Loiacono2,3, Maja Cemazar1,4, Urska Kamensek1, Gregor Sersa1, Emanuela

Signori2,5

1Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana, Slovenia 2Laboratory of Genetic and Clinical Pathology, University Campus Bio-Medico of Rome, via Álvaro

del Portillo 21, 00128 Rome, Italy. 3Laboratory of Oncology, IRCCS “Casa Sollievo della Sofferenza”, viale dei Cappuccini, 71013 San

Giovanni Rotondo (Fg), Italy. 4Faculty of Health Sciences, University of Primorska, Polje 42, Izola, Slovenia 5National Research Council - Institute of Translational Pharmacology (CNR-IFT), Via Fosso del Cavaliere 100, Rome, Italy

ABSTRACT

The use of plasmids encoding interleukin-12 (IL-12) in gene electrotransfer studies has shown great

promise in treating tumors induced in experimental animals, and in human and veterinary clinical

studies1. These studies were using plasmids carrying the gene for antibiotic resistance, which is not

recommended for human therapy. Therefore, the aim of this study was to construct an antibiotic-free

plasmid and to evaluate its antitumor effectiveness in murine B16F10 melanoma. The antibiotic-free

plasmid encoding IL-12 (pORFmIL-12ORT) was successfully constructed using ORT technology (Cobra

bio, Keele, UK). Gene electrotransfer of pORFmIL-12ORT was performed in B16F10 tumors growing

subcutaneously in C57Bl/6 mice. The antitumor effectiveness was evaluated by tumor growth delay

assay and local immune response was detected by hemotoxylin eosin (HE) staining and

immunohistochemical (IHC) staining of F4/80 and MHCII. Systemic effect was evaluated by the IL-12

and IFN determination in blood. All the IL-12 treated tumors responded completely to the therapy by

the day 14. HE staining showed infiltration of immune cells, which lasted from day 4 to day 14. IHC

staining for F4/80 and MHCII showed higher intensity of positive cells at day 4, which lasted until day

14, compared to control. Serum IL-12 and IFN showed increased levels of IL-12 at days 1 and 4 and of

IFN at days 4 and 8. Our results showed complete tumor response after gene electrotransfer of

pORFmIL-12ORT and increased immune cells infiltration into the tumors. Also, high infiltration of

MHCII positive cells in the IL-12 treated tumors proposes a M1 polarization of macrophages, that is

important for the tumor elimination2.

REFERENCES 1. Cemazar M, Jarm T, Sersa G. Cancer electrogene therapy with interleukin-12. Curr Gene Ther. 2010, 10(4), 300 - 11

2. Chanmee T, Ontong P, Konno K, Itano N. Tumor-Associated Macrophages as Major Players in the Tumor

Microenvironment. Cancers. 2014, 6(3), 1670 - 90


26 | P a g e

Corresponding author: Katja Uršič ([email protected])

Abstract

PERITUMORAL GENE ELECTROTRANSFER OF IL-12 AS ADJUVANT IMMUNOTHERAPY TO

INTRATUMORAL ELECTROCHEMOTHERAPY WITH CISPLATIN FOR TREATMENT OF MURINE

B16F10 MELANOMA

Katja Uršič1, Špela Kos1, Urška Kamenšek1, Maja Čemažar1,2, Gregor Serša1

1Institute of Oncology Ljubljana, Department of Experimental Oncology, Slovenia

2University of Primorska, Faculty of Health Sciences, Slovenia

ABSTRACT

Treatment of metastatic melanoma has undergone rapid renovation, especially in the light of

combining established therapies with recently discovered immunotherapies. Our research group has

been studying intratumoral electrochemotherapy (i.t. ECT; a combination of chemotherapeutic drug

and electric pulses) as a local ablative therapy which is a well-established therapy in preclinical models

as well as in clinics.1 ECT with cisplatin (CDDP) results not only in direct cytotoxic effect on cancer cells,

but also indirectly affects the immune system.2,3 However, after applying low-dose i.t. ECT with CDDP

(2 mg/kg) to murine B16F10 melanoma, we observed only a delayed growth of primary tumors with

no complete responses. To achieve both better local tumor growth control and an effect on distant

non-treated metastasis (abscopal effect), we applied peritumoral gene electrotransfer (p.t. GET) of

plasmid encoding interleukin-12 (IL-12) as an adjuvant immunotherapy.4 IL-12 is a proinflammatory

cytokine which monitors inflammation via innate and adaptive immune system responses.5 With p.t.

GET of therapeutic plasmid encoding mouse IL-12 under constitutive EF-1α/HTLV hybrid promoter, we

achieved low dose expression of the transgene in surrounded (p.t.) skin tissue, without any systemic

cytotoxicity. Treatment by p.t. GET (50 µg/tumor) alone resulted in only tumor growth delay but no

complete responses, whereas with combined therapy (i.t. ECT with CDDP and p.t. GET with IL-12), we

cured 37.5% of animals. Further, we also observed abscopal effect in combined therapy. Thus we

conclude that p.t. GET of IL-12 potentiates the effect of i.t. ECT with CDDP on local and systemic level.

REFERENCES 1. Yarmush ML, Golberg A, Serša G, Kotnik T, and Miklavčič D. Electroporation-Based Technologies for Medicine: Principles,

Applications, and Challenges. Annu. Rev. Biomed. Eng. 2014, 16, 295–320.

2. Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer. 2007, 7, 573–584.

3. Hato SV, Khong A, De Vries IJM, and Lesterhuis WJ. Molecular pathways: The immunogenic effects of platinum-based

chemotherapeutics. Clin. Cancer Res. 2014, 20, 2831–2837.

4. Sersa G, Teissie J, Cemazar M, Signori E, Kamensek U, Marshall G, and Miklavcic D. Electrochemotherapy of tumors as in

situ vaccination boosted by immunogene electrotransfer. Cancer Immunol. Immunother. 2015, 64, 1315–1327.

5. Tugues S, Burkhard SH, Ohs I, Vrohlings M, Nussbaum K, Vom Berg J, Kulig P, and Becher B. New insights into IL-12-

mediated tumor suppression. Cell Death Differ. 2015, 22, 237–246.


27 | P a g e

Corresponding author: Katarina Žnidar, [email protected]

Abstract

THE EFFECT OF GENE ELECTROTRANSFER OF BLANK pDNA ON TUMOR CELLS' SURVIVAL

AND EXPRESSION OF CYTOSOLIC DNA SENSORS

Katarina Žnidar1, Maša Bošnjak2, Nina Semenova3, Loree Heller3, 4, Maja Čemažar1, 2

1University of Primorska, Faculty of Health Sciences, Slovenia 2Institut of Oncology Ljubljana, Slovenia 3Old Dominion University, Frank Reidy Research Center for Bioelectrics, USA 4Old Dominion University, College of Health Sciences, USA

ABSTRACT

Gene electrotransfer (GET) is an effective non-viral delivery method for introducing plasmid DNA

(pDNA) into cells and tissues. After exposure of cells to the electric pulses, pDNA enters the cells and

reaches the cytosol via endocytosis or through electroporation induced pores [1]. Preclinical studies

showed tumor growth delay and regression after GET of the blank pDNA, however the possible

mechanisms responsible for tumor regression are not known [2,3]. One of the possible mechanism

could be the activation of DNA-specific receptors in the cytosol referred to as cytosolic DNA sensors.

Their response to DNA leads to upregulation of interferons and cytokines as well as cell death [4].

In our study, the presence of cytosolic DNA sensors in different mouse tumor cells (B16F10, TS/A, WEHI

164) were demonstrated using RT-PCR and western blot. After GET of control pDNA several DNA

sensors (DDX60, DAI, p204) and Interferon β (IFN β) were upregulated on mRNA and protein level.

pDNA concentration-dependent decrease in cell survival after pDNA GET was obtained in all cell lines,

with TS/A cell line the least susceptible. After GET of control pDNA, a higher number of necrotic cells

were observed in two cell lines (B16F10 and WEHI 164) compared to TS/A cells as determined by flow

cytometry using annexin V and 7AAD assay to characterize apoptotic or necrotic cell death.

To conclude, the increased expression of several DNA sensors and IFNβ after GET of blank pDNA

indicate that this might be the possible mechanism for observed cell death of tumor cells.

REFERENCES 1. Rosazza C, Phez E, Escoffre JM, Cezanne L, Zumbusch A, and Rols MP. Cholesterol implications in plasmid DNA

electrotransfer: Evidence for the involvement of endocytotic pathways. Int J Pharm 2012, 423: 134-143.

2. Heller L, Todorovic V, Cemazar M. Electrotransfer of single-stranded or double-stranded DNA induces complete

regression of palpable B16.F10 mouse melanomas. Cancer Gene Ther 2013, 20:695-700.

3. Ugen KE, Kutzler MA, Marrero B, Westover J, Coppola D, Weiner DB, et al. Regression of subcutaneous B16 melanoma

tumors after intratumoral delivery of an IL-15-expressing plasmid followed by in vivo electroporation. Cancer Gene Therapy

2006, 13: 969-974.

4. Desmet CJ, Ishii KJ . Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination. Nat Rev

Immunol 2012, 12:479-491.


28 | P a g e

Corresponding author: Vasja Progar, [email protected]

Abstract

FUNCTIONAL ENRICHMENT OF DIFFERENTIALLY EXPRESSED GENES IN HOP INFECTED BY V.

nonalfalfae

Vasja Progar1, Sabina Berne1, Jernej Jakše1, Nataša Štajner1, Sebastjan Radišek2, Branka Javornik1

1University of Ljubljana, Biotechnical Faculty - Department of Agronomy, Slovenia 2Slovenian Institute for Hop Research and Brewing, Plant Protection Department, Slovenia

ABSTRACT

Hop (Humulus lupulus L.) is an agriculturally important plant with uses in brewing and pharmacy. Its

production is hampered by Verticillium wilt, predominantly caused by a soil-borne pathogenic fungus

Verticillium nonalfalfae. Some hop cultivars exhibit resistance against this disease but underlying

molecular mechanisms of resistance remain to be determined.

In this study, resistant and susceptible hop plants were infected with a lethal strain of V. nonalfalfae.

RNA-Seq was performed on roots and shoots of infected and control plants harvested at 6, 12, 18 and

30 days post inoculation.

Sequenced reads were mapped to the gene models of hop draft genome1, which were then assigned

Arabidopsis gene IDs based on sequence similarity. Differentially expressed genes (DEGs) were

determined using FunPat2 and subjected to functional network analysis using ClueGO3.

The susceptible hop cultivar mounted a strong basal defence response against V. nonalfalfae, with

enhanced chitinase and oxidoreductase activity and downregulation of photosynthetic processes in

shoots. In the resistant cultivar, cutin metabolism and cell wall biogenesis were enriched in DEGs in

shoots and terpenoid biosynthesis and polysaccharide catabolism in roots. Additionally, the network

analysis exposed several DEGs at intersections of the enriched functional groups. These potentially

code for hub proteins coordinating the hop response to V. nonalfalfae and identification of their exact

roles in this pathosystem should be pursued in further studies.

REFERENCES 1. HopBase v1.0. http://hopbase.org/ 2. Sanavia T., Finotello F., Di Camillo B. FunPat: function-based pattern analysis on RNA-seq time series data. BMC Genomics

2015, 16(Suppl 6), S2. 3. Bindea G., Mlecnik B., Hackl H., Charoentong P., Tosolini M., Kirilovsky A., Fridman W.H., Pages F., Trajanoski Z., Galon J.

ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. 2009.

Bioinformatics 2009, 25(8), 1091-1093.


29 | P a g e

Corresponding author: Kristina Marton, [email protected]

Abstract

Verticillium nonalfalfae AND ITS CANDIDATE SECRETED EFFECTOR PROTEINS

Marton Kristina1, Berne Sabina1, Progar Vasja1, Štajner Nataša1, Flajšman Marko1, Radišek Sebastjan2,

Javornik Branka 1

1Biotechnical faculty, Department of Agronomy, Slovenia 2Slovenian Institute for Hop Research and Brewing, Plant protection department, Slovenia Country

ABSTRACT

Verticillium nonalfalfae (Vna)1 is a soil born phytopathogenic ascomycete that causes verticillium

wilting on a wide range of crops, including hop. Apart from phytosanitary measures, the only effective

approach is planting resistant varieties. In modern resistance breeding programs, plant pathogen

effectors are exploited as a tool to detect resistance (R) genes and functionally characterize R proteins

that recognize different effectors2. Effectors are small secreted proteins that modulate host immune

responses and can also serve as avirulence factors. We report here an in-house pipeline for effector

detection of phytopathogenic fungi.

The putative Vna proteome containing 9,269 gene models was functionally annotated with a wide

range of analyses using blast and hmmer searches against different databases. Based on signal peptide,

transmembrane domain predictions and putative cellular localization, we determined the Vna

secretome, comprising 962 gene models. Three hundred and thirty CSEPs (candidate secreted effector

proteins) were identified after omitting gene models with carbohydrate-active enzyme annotation and

retaining gene models with none or only effector-specific PFAM domains. For the 49 best ranked CSEPs

(based on in planta expression, transcriptomic data of differentially expressed gene models, no

sequence homology to other known genes, lineage-specificity for highly virulent Vna etc.), we

examined their temporal and spatial expression during hop infection using RT-qPCR and identified the

six best effector candidates. Two of them were knocked out from Vna conidia, which were used for

inoculation of susceptible hop plants. Disease symptoms evaluation revealed reduced virulence of

knock outs.

REFERENCES 1. Inderbitzin P, Bostock RM, Davis RM, Usami T, Platt HW, Subbarao K V. Phylogenetics and taxonomy of the fungal

vascular wilt pathogen Verticillium, with the descriptions of five new species. PLoS One. 2011, 6(12), e28341.

2. Klosterman SJ, Subbarao K V, Kang S, et al. Comparative genomics yields insights into niche adaptation of plant vascular

wilt pathogens. Dangl JL, ed. PLoS Pathog. 2011, 7(7), e1002137.


30 | P a g e

Corresponding author: Marija Rogar, [email protected]

Abstract

POLYMORPHISMS IN SEGREGATION GENES INVOLVED IN THE DEVELOPMENT OF GASTRIC

CANCER IN SLOVENIAN POPULATION

Marija Rogar1, Petra Hudler1, Radovan Komel1

1 Medical Centre for Molecular Biology, Faculty of Medicine UL, Vrazov trg 2, 1000 Ljubljana

ABSTRACT

Gastric cancer is very complex and heterogeneous disease, and while numerous studies focusing on its

development were carried out, the detailed mechanisms still remain unclear1. Genomic instability is

one of the main topics of gastric cancer genetic studies2. Single nucleotide polymorphisms (SNPs) in

genes encoding mitotic kinases as well as of other segregation genes, or their combinations, may result

in the development of cancer3.

Polymorphisms were determined using quantitative real-time PCR (qPCR), restriction fragment length

polymorphism (RFLP) and DNA sequencing.

The association between polymorphisms rs2277559 (BUB1B), rs2241666 (ZWINT), rs11858113 (CASC5)

and rs11855334 (CASC5) and increased risk of developing gastric cancer in male population was

determined. Genotypes of six polymorphisms (in BUB1B gene) rs2277559, rs2290551, rs1801376,

rs1047130, rs1565866, rs2277560 were associated with Lauren classification. On the other hand, the

link between genotypes of polymorphisms rs1801376 (BUB1B), rs11855334 (CASC5), rs2241666

(ZWINT), rs2910101 (PTTG1) and rs1047130 (BUB1B) and tumour differentiation was observed.

Survival analysis revealed association between the lymph node involvement and perineural invasion.

Statistically higher frequencies of haplotypes G-A-G-T-G-G-A, G-G-A-G-A-A-G and A-G-G-T-A-G-A in

gene BUB1B and of haplotypes A-A-A-C and C-C-G-T in gene ESPL1 were observed in gastric cancer

patients, while haplotypes A-C-A-T and C-A-G-T in gene ESPL1 were found frequently present in the

group of controls.

Specific genotypes of selected polymorphisms in chromosome segregation genes were found

associated with development of gastric cancer in Slovenian population of gastric cancer patients.

Polymorphisms linked to gastric cancer could serve as potential diagnostic and prognostic biomarkers.

REFERENCES 1. Hudler P.: Genetic aspects of gastric cancer instability. The Scientific World Journal. 2012; 2012: 761909.

2. Lengauer C., Kinzler K. W., Vogelstein B.: Genetic instability in colorectal cancers. Nature. 1997; 386(6625): 623-7.

3. Frank S. A.: Genetic predisposition to cancer - insights from population genetics. Nature reviews Genetics. 2004;

5(10): 764-72.


31 | P a g e

Corresponding author: Alja Zottel, [email protected]

Abstract

GLIOBLASTOMA MULTIFORME AND NANOBODIES

Alja Zottel1, Ivana Jovčevska1, Neja Zupanec1, Radovan Komel1

1 Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana

ABSTRACT

Glioblastoma multiforme (GBM) is an astrocytoma type of brain cancer and is one of the deadliest

cancers.1 In spite of the standard therapy that includes surgery, radiotherapy and chemotherapy, most

of the patients do not survive more than two years.1 There are many reasons why the therapies fail.

One of the biggest reasons is the existence of glioblastoma stem cells that are resistant to

chemotherapy and radiotherapy and are able to form a new tumor.2 The other reason is that it is

difficult to surgically remove tumor completely.3 One of most promising devices for targeting GBM are

nanobodies, small antigen-recognizing part of heavy chain antibody, that is mostly found in llamas,

camels and sharks.4 The are many advantages of nanobodies regarding classical antibodies such as high

yield in production, low immunogenicity, solubility and stability at high temperature as extreme pH.4,5

The aim of our work was to evaluate the effect of four nanobodies on U87MG, U251MG and NCH

glioblastoma cells lines. Furthermore, we have determined the location of specific antigens using FITC

bound nanobodies and have verified our results with commercial antibodies. We confirmed the results

for three nanobodies and one has to be confirmed in future. Nanobodies for different antigens effect

cells in different way. We conducted two different metabolic assays: WST test and AlamarBlue test.

The results show that the most cytotoxic for NCH cells is Nb206 and for U87MG and U251MG are the

most cytotoxic Nb10, Nb79 and Nb206 nanobodies.

REFERENCES

1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant

temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987-96.

2. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential

activation of the DNA damage response. Nature. 2006;444(7120):756-60.

3. Van Meir EG, Hadjipanayis CG, Norden AD, Shu HK, Wen PY, Olson JJ. Exciting new advances in neuro-oncology: the avenue

to a cure for malignant glioma. CA Cancer J Clin. 2010;60(3):166-93.

4. Deffar K, Shi H, Li L, Wang X, Zhu X. Nanobodies - the new concept in antibody engineering. African Journal of Biotechnology.

2009;8(12):2645-52

5. Oliveira S, Heukers R, Sornkom J, Kok RJ, van Bergen En Henegouwen PM. Targeting tumors with nanobodies for cancer

imaging and therapy. J Control Release. 2013;172(3):607-17.


32 | P a g e

POSTERS


33 | P a g e

ABSTRACTS and PAPERS of POSTERS

MOLECULAR BASIS OF DISEASES

Marko Vidak, Damjana Rozman, Mirjana Liović, Radovan Komel

Search of new glioblastoma stem cell markers by comparing microarrays data from the

NCBI GEO database, and experimental validation of those markers in glioma cell lines

Matej Rebek, Eva Jakljevič, Darja Žgur-Bertok, Marjanca Starčič Erjavec

Phylogenetic groups and clbAQ, usp, hlyA, cnf1 genes among commensal escherichia

coli strains isolated from feces of healthy humans of both genders and both age groups

Tinkara Prijatelj, Danijela Vočanec, Tadej Pajič, Martina Fink, Irena Preložnik Zupan, Peter

Černelč, Radovan Komel, Tanja Kunej, Nataša Debeljak

Novel molecular-genetic diagnostic test for JAK2 negative erythrocytosis

Rok Končnik, Barbara Krajnc, Jovan Krsteski, Uroš Potočnik

Polymorphism rs1056837 in CYP1B1 encoding an estrogen metabolizing enzyme

presents a risk for ULM among Slovenian women

Matjaž Deželak, Žan Hribar, Carina E. P. Kozmus, Uroš Potočnik

A polymorphism located in 3’UTR of PITHD1 near cannabinoid receptor 2 gene CNR2

is associated with severe forms of Crohn’s disease and CNR1 gene expression


34 | P a g e

Corresponding author: Marko Vidak ([email protected])

Abstract

SEARCH OF NEW GLIOBLASTOMA STEM CELL MARKERS BY COMPARING MICROARRAYS

DATA FROM THE NCBI GEO DATABASE, AND EXPERIMENTAL VALIDATION OF THOSE

MARKERS IN GLIOMA CELL LINES

Marko Vidak1, Damjana Rozman1 , Mirjana Liović1 and Radovan Komel1

1University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, Vrazov Trg 2, SI-1000 Ljubljana, Slovenia

ABSTRACT

Glioblastoma multiforme (GBM) is the most lethal brain tumor. Glioblastoma stem cells are believed

to be the reason for the tumor's malignancy. Identification of stem cells among other cells in the tumor

mass would therefore facilitate GBM therapy. Most likely there are different groups of stem cells

within a single tumor, each such group expressing its own set of markers1. Our goal is to find new

markers that would enable targeting of stem cell populations not expressing any of already known

markers.

Gene expression profiles of glioblastoma cell lines will be compared with the profiles of non-malignant

brain cells from the same dataset. All the profiles have been obtained from microarrays and uploaded

to the NCBI GEO database. The goal is to find genes that are significantly overexpressed in malignant

cells in most or all the analyzed datasets. The proteins coded by these genes will be assessed by their

function, intracellular location and expression frequency in brain and other tissues. Most interesting

candidates, i.e. surface proteins that are not too commonly expressed outside the brain and whose

function is related to growth and proliferation, will have their expression investigated in the U87

glioma cell line and the NCH CD133+ purported glioblastoma stem cell line, as well as in a new cell line

derived from the U87. Compared to the original U87 line, this new cell line is believed to be enriched

in stem-cell like cells, including CD133- stem cells not present in the NCH line.

REFERENCES 1. Wang J, Sakariassen P, Tsinkalovsky O, Immervoll H, Bøe SO, Svendsen A, et al. CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. Int J Cancer. 2008, 122(4), 761-768.


35 | P a g e

Corresponding author: Matej Rebek ([email protected]):

Article

PHYLOGENETIC GROUPS AND clbAQ, usp, hlyA, cnf1 GENES AMONG COMMENSAL

Escherichia coli STRAINS ISOLATED FROM FECES OF HEALTHY HUMANS OF BOTH GENDERS

AND BOTH AGE GROUPS

Matej REBEK1, Eva JAKLJEVIČ2, Darja ŽGUR-BERTOK1, Marjanca STARČIČ ERJAVEC1

1Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia 2Grammar school Poljane, Strossmayerjeva 1, 1000 Ljubljana, Slovenia

The bacterium E. coli and its host usually coexist in mutual benefit; E. coli is known to be an important member of the gut

microbiota. But there are several E. coli strains that have specific virulence factors and can cause a variety of different

intestinal and extraintestinal infections. It is known that the gut microbiota can be the reservoir of such strains. The aim

of our study was to reveal if carriage of such potential pathogenic E. coli strains in the gut differs among females and

males and among children (0-18 years) and adults (>18 years). To fulfill this aim we isolated on MacConkey plates E. coli

from feces of healthy humans of both genders and both age groups. To confirm the identification of E. coli the indole

test, URISELECT plates and LAMP assay were used. ERIC-PCR was performed to distinguish different isolates/strains. We

obtained a collection of 56 different E. coli strains. All strains were screened for phylogenetic groups by the triplex PCR as

well as the extended quadruplex PCR phylotyping method and the following virulence-associated genes: clbA, clbQ, cnf1,

hlyA and usp. The results showed that around 50% of all tested strains belonged to the phylogenetic group B2. The genes

clbA, clbQ were detected in 62%,usp in 55%, hlyA in 20% and cnf1 in 18% of all tested E. coli strains. There was no

statistical significant difference in carriage of potential pathogenic E. coli strains among females and males and among

children and adults. Our results showed that in the intestine of healthy humans of both genders and age groups there is a

high virulence potential for extraintestinal pathogenicE. coli strains.

INTRODUCTION

Escherichia coli (E. coli) is the commensal species of the facultative anaerobic microbiota in the lower

intestine of healthy warm-blooded organisms. It colonizes the gastrointestinal tract of infants within

a few hours after birth and mostly coexists with its host in mutual benefit for decades1. However, it is

assumed that the intestinal microbiota is also a reservoir of the so called extraintestinal pathogenic E.

coli (ExPEC) strains that can, due to specific virulence factors instigate an impressive variety of

extraintestinal infections2. Characteristic virulence traits that are present in most ExPEC include

various adhesins, factors to avoid or subvert host defence systems (e.g. capsule, Toll/interleukin-1

receptor domain–containing protein), mechanisms for nutrient acquisition (e.g. siderophores), and

toxins (e.g. hemolysin, cytotoxic necrotizing factor 1)3. It is known that ExPEC mainly belong to

phylogenetic group B2 and to a lesser extent to the group D. In addition, many virulence factors are

associated with the B2 group3, 4.

Yet, data on the prevalence of potential ExPEC strains among the bowel microbiota from healthy

humans are very limited2. To further our understanding of the potential of commensal E. coli to

cause extraintestinal infections and to compare their virulence potential in relation to host

gender/age we screened a collection of 56 E. coli strains, isolated from feces of healthy humans of

both genders and both groups (0-18 and >18), for the prevalence of several extraintestinal virulence-

associated genes. Statistical analysis was performed to reveal a possible difference in ExPEC carriage

among females and males and both age groups.


36 | P a g e

METHODS

E. coli strains were isolated as lactose-positive colonies on MacConkey agar plates from feces of 56

individuals. The indole test was performed to ascertain the E. coli species. The investigated strains

were cultivated in Luria Bertani medium or agar at 37˚C.

We used ERIC-PCR to differentiate E. coli isolates/strains. The ERIC-PCR profiles, following agarose gel

electrophoresis, were determined manually. We used the same method as described in Fajs et al.,

20135. Prior to profiling, boiled lysates from isolates were prepared6 to be used in PCR reactions.

To verify the identification of E. coli we used the LAMP assay. LAMP assay method was performed as

described in Hill et al., 20087. The lysates, as well as PCR reactions, were prepared in duplicates.

E. coli strains were screened for phylogenetic groups with the triplex PCR method differentiating the

phylogenetic groups A, B1, B2, D8 and with the extended quadruplex PCR method differentiating the

phylogenetic groups A, B1, B2, C, D, E, F9. We also performed a screening for the following virulence-

associated genes: clbAQ, cnf1, hlyA and usp. The primers and PCR programs used were described

previously3, 10, 11, 12, 13. The lysates, as well as PCR reactions, were prepared in duplicates.

To analyze the data we used Fisher's exact test (two-tailed). The threshold for statistical significance

was set at p values of < 0.05.

RESULTS

ERIC-PCR was performed on all 56 isolates. ERIC-PCR produced profiles with bands ranging from less

than 250 bp to approximately 3000 bp. Based on analysis of ERIC-PCR profiles, all the isolates had

different ERIC-PCR profiles and were thus considered genetically distinct.

Products of the LAMP assay were analyzed with agarose gel electrophoresis (Figure 1) and we were

able to verify the identification of E. coli in all but two isolates. These isolates were not E. coli,

therefore we excluded them from this research.

Figure 1: Agarose gel electrophoresis of LAMP products. LAMP products were analyzed on a 1% agarose gel. 1 – GeneRulerTM 50 bp DNA ladder (Fermentas, St. Leon-Rot, Germany); 2,3 – isolate 2001 ž; 4,5 – isolate 1999ž/8; 6,7 – not E. coli; 8,9 – isolate 1998ž/2; 10,11 – isolate 1999ž/5; 12,13 – isolate 1999ž/4; 14, 15 – isolate 1969m; 16 – negative control.

Our analysis showed that the majority of the strains, studied with the triplex PCR method1, belonged

to groups B2 and A, 27 (48%) were assigned to the group B2 and 11 (20%) were assigned to group A.

Ten (18%) isolates were grouped as D and 8 (14%) belonged to the B1 group (Figure 2). As expected,

when tested with the extended quadruplex PCR method2, the results were quite similar. Twenty-six


37 | P a g e

(46%) of the studied strains belonged to the group B2, 14 (25%) of them belonged to the group A and

8 (14%) of the strains belonged to the group B1. Only a few of the strains belonged to groups C, D, E

and F (Figure 2).

Figure 2: Prevalence of E. coli strains by phylogenetic groups using the triplex PCR method (A) and the extended quadruplex PCR method (B).

The most prevalent virulence-associated genes among the studied strains were clbAQ, which was

found in 35 (62%) of the tested strains, followed by usp, detected in 31 (55%) strains, cnf1 was found

in 10 (18%) strains and hlyA in 52 (58%) strains (Figure 3).

Figure 3: Prevalence of virulence-associated genes, found among studied E. coli strains

Analysis of the distribution of virulence-associated genes in relation to age revealed that no

statistically significant differences were found between the age group 0-18 years and the age group

>18 years (Table 1).


38 | P a g e

Table 1: Positive E. coli strains for virulence-associated genes in relation to age

No. (%) of positive strains

0–18 years >18 years p value1

(n = 27) (n = 29)

Virulence genes

clbAQ 20 (74) 15 (52) 0.104

cnf1 5 (19) 5 (17) 1

hlyA 6 (22) 5 (17) 0.742

usp 17 (63) 14 (48) 0.296 1 Calculated by Fisher’s exact test.

The performed analysis of the distribution of virulence-associated genes in relation to gender

showed that the studied traits were rather equally distributed among the E. coli isolates from males

and females (Table 2).

Table 2: Positive E. coli strains for virulence-associated genes in relation to gender

No. (%) of positive strains

Female Male p value1

(n = 41) (n = 15)

Virulence genes

clbAQ 25 (61) 10 (67) 0.764

cnf1 9 (22) 1 (7) 0.259

hlyA 10 (24) 1 (7) 0.255

usp 21 (51) 9 (60) 0.763 1 Calculated by Fisher’s exact test.

DISCUSSION

As our analysis showed that more than 60% of the studied isolates belonged to either the B2 or D

phylogenetic groups when studied with triplex PCR method8, and almost 50% of isolates belonged to

the B2 phylogenetic group when studied with extended quadruplex PCR method9, it can be assumed

that the majority of the tested isolates exhibit a high virulence potential4. Accordingly, a high

prevalence of several virulence-associated genes associated with the B2 group was detected. Thus,

many of the studied strains harbored genes characteristic of ExPEC. Due to morphological differences

and differences in dynamics (transit time), related to gender as well as age14, 15, we expected to find

several statistically significant associations in relation to host gender and age. However, in our study,

among isolates from males and females no statistically significant differences were found (Table 1

and Table 2). To conclude, the review article Starčič Erjavec and Žgur-Bertok, 201516 showed that

there is high virulence potential for extraintestinal infections among commensal E. coli isolated from

healthy humans and our study showed that healthy females and males and both age groups have no

statistical significant difference in carriage of potential ExPEC.


39 | P a g e

REFERENCES

1. Kaper J, Nataro J, Mobley H. Pathogenic Escherichia coli.Nat. Rev. Microbiol. 2004, 2, 123–140.

2. Tenaillon O, Skurnik D, Picard B, Denamur E. The population genetics of commensal Escherichia coli. Nat. Rev. Microbiol.

2010, 8, 207–217.

3. Russo T, Carlino U, Johnson J. Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic

isolate of Escherichia coli. Infect. Immun. 2001, 69, 6209–6216.

4. Picard B, Garcia J, Gouriou S, Duriez P, Brahimi N, Bingen E, Elion J, Denamur E. The link between phylogeny and virulence

in Escherichia coli extraintestinal infection. Infect. Immun. 1999, 67, 546–553.

5. Fajs L, Jelen M, Borić M, Đapa T, Žgur-Bertok D, Starčič-Erjavec M. The discriminative power in determining genetic

diversity of Escherchia coli isolates: Comparing ERIC-PCR with AFLP. Afr. J. Microbiol. Res. 2013, 70, 20, 2416-2419.

6. Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesin-encoding

operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J. Clin. Microbiol. 2009, 30, 1189–1193.

7. Hill J, Beriwal S, Chandra I, Paul V, Kapil A, Singh T, Wadowsky R, Singh V, Goyal A, Jahnukainen T, Johnson J, Tarr P, Vats

A. Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J. Clin. Microbiol.

2008, 46, 8, 2800-2804.

8. Clermont O, Bonacorsi S, Bingen A. Rapid and simple determination of the Escherichia coli phylogenetic group. App.

Environ. Microbiol. 2000, 66, 10, 4555-4558.

9. Clermont O, Christenson J, Denamur E, Gordon D. The Clermont Escherichia coli phylo-typing method revisited:

improvement of specificity and detection of new phylo-groups. Environ. Microbiol. Rep. 2013,5, 1, 58-65.

10. Johnson J, Russo T, Tarr P, Carlino U, Bilge S, Vary Jr. J, Stell A. Molecular epidemiological and phylogenetic associations

of two novel putative virulence genes, iha and iroN(E. coli), among Escherichia coli isolates from patients with urosepsis,

Infect. Immun. 2000, 68, 3040–3047.

11. Johnson J, Stell A, Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to

phylogeny and host compromise., J. Infect. Dis. 2000,181, 261–272.

12. Schubert S, Rakin A, Karch H, Carniel E, Heesemann J. Prevalence of the "high-pathogenicity island" of Yersinia species

among Escherichia coli strains that are pathogenic to humans. Infect. Immun. 1998, 66, 480–485.

13. Starčič Erjavec M, Arbiter T, Žgur-Bertok D. Pathogenicity islands, plasmids and iron uptake systems in extraintestinal

pathogenic Escherichia coli strains. Acta biol. slov. 2009, 52, 73–83.

14. Graff J, Brinch K, Madsen J. Gastrointestinal mean transit times in young and middle-aged healthy subjects. Clin. Physiol.

2001, 21, 253–259.

15. Hounnou G, Destrieux C, Desmé J, Bertrand P, Velut S. Anatomical study of the length of the human intestine. Surg.

Radiol. Anat. 2002, 24, 290–294.

16. Starčič Erjavec M, Žgur-Bertok D. Virulence potential for extraintestinal infections among commensal Escherichia coli

isolated from healthy humans – the Trojan horse whitin our gut. FEMS Microbiol. Lett. 2015, 362, 1-9.


40 | P a g e

Corresponding author: Prof.dr. Nataša Debeljak, [email protected]

Abstract

NOVEL MOLECULAR-GENETIC DIAGNOSTIC TEST FOR JAK2 NEGATIVE ERYTHROCYTOSIS

Tinkara Prijatelj1,2 & Danijela Vočanec1,2, Tadej Pajič3, Martina Fink3, Irena Preložnik Zupan3, Peter

Černelč3, Radovan Komel1, Tanja Kunej2 and Nataša Debeljak1

¹ Institute of Biochemistry, Faculty of Medicine, University of Ljubljana

² Department of Animal Science, Biotechnical Faculty, University of Ljubljana

³ Clinical department of Haematology, University Medical Centre Ljubljana

ABSTRACT

Erythrocytosis is heterogeneous group of disorders characterized by the expansion of the erythrocyte

compartment including elevated red blood cell (RBC) number, haematocrit, and haemoglobin content

in the peripheral blood. Familial erythrocytosis (FE) is a group of rare congenital disorders with various

genetic background. Erythropoietin receptor gene (EPOR) mutations are the indicator for primary

familial erythrocytosis. Secondary erythrocytosis syndromes are typically associated with a defect in

various genes included in oxygen sensing pathway that leads to the increased erythropoietin

production (1). Current diagnostic procedure in Slovenia enables exclusion of JAK2 gene mutations,

the cause of polycythaemia vera (PV). Our study was the basis of new molecular-genetic test for EPO

and EPOR genes in JAK2 negative erythrocytosis.

Sequence analysis of EPO promoter and 3’ enhancer and EPOR exon 8 was performed on two related

patients with erythrocytosis with unknown cause.

Primary familial erythrocytosis due to EPOR mutation was excluded by sequence analysis in both

patients. So far, 24 mutations in EPOR, located in exon 8, have been associated with erythrocytosis.

Mutations are leading to cytoplasmic truncation of the receptor and loss of the C-terminal negative

regulatory domain (2).

However, sequence analysis revealed mutation in 3’ enhancer region of the EPO gene in both patients,

previously described in blood donors with upper limit haematocrit. The role of erythropoietin in

erythrocytosis is indirect and previously had not been linked to the disease.

New molecular-genetic test for analysis of the EPO (promoter and 3’ enhancer) and EPOR (exon 8)

mutations was successfully implemented in clinical use.

REFERENCES

1. G. Lee, M. O. Arcasoy, The clinical and laboratory evaluation of the patient with erythrocytosis. Eur J Intern Med 26,

297-302 (2015).

2. C. Bento et al., Genetic basis of congenital erythrocytosis: mutation update and online databases. Hum Mutat 35,

15-26 (2014).


41 | P a g e

Corresponding author: Rok Končnik, [email protected]

Abstract

POLYMORPHISM rs1056837 IN CYP1B1 ENCODING AN ESTROGEN METABOLIZING ENZYME

PRESENTS A RISK FOR ULM AMONG SLOVENIAN WOMEN

Rok Končnik1, Barbara Krajnc1, Jovan Krsteski2, Uroš Potočnik2, 3

1Faculty of Medicine, University of Maribor, Slovenia 2Center for Human Genetics and Pharmacogenetics, Faculty of Medicine, University of Maribor, Slovenia 3Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty for Chemistry and Chemical Engendering, University

of Maribor, Slovenia

ABSTRACT

Uterine leiomyomas (ULM) are the most common benign uterine neoplasms affecting a majority of

reproductive aged women1. The aetiology of ULM is poorly understood, especially the molecular bases

for these tumors. Several studies suggest that ULM are influenced by genetic risk factors2, 3, 4. One of

the molecular mechanisms that may be involved are polymorphisms of genes involved in estrogen

synthesis and/or metabolism (CYP1A1, CYP2A13, CYP1B1 and COMT) 2, 5.The aim of our study was to

investigate whether polymorphisms in the selected genes, coding for CYP1A1 (rs1799814), CYP2A13

(rs13809886), CYP1B1 (rs1056837) and COMT (rs4680) are associated with ULM susceptibility in

Slovenian women with solitary and multiple leiomyomas.

We investigated 180 women with clinically diagnosed ULM and 134 controls from Slovene population.

Genotypization of rs1799814, rs13809886, rs1056837 and rs4680 was performed using polymerase

chain reaction followed by either restriction fragment length polymorphism or high-resolution melting

analyses. When examining genotype frequencies of all patients and general control, AA genotype of

rs1056837 was associated with ULM susceptibility compared to AG and GG genotypes in dominant

genetic model (p = 0.0001; OR = 3.315; 95% CI 1.788-6.146). Conversely, in recessive genetic model,

GG genotype was associated with ULM susceptibility compared to AG and AA genotypes (p = 0.026;

OR = 2,033; 95% CI = 1.099-3.761). Analysis of associations between SNPs and clinical data within

particular ULM patient group revealed that multiple ULM patients with AA genotype of were more

likely to have previous inflammation of genital tract (p = 0.002; OR = 0.0262; 95% CI = 0.112-0.613).

There were no statistically significant associations of other polymorphisms.

In our study we noted a significant difference between patients with ULM and general subjects in

CYP1B1 rs10568 regarding expression of AA and GG genotypes. Results suggest co-dominant influence

of alleles A and G on ULM phenotype where only a combination of both alleles have protective function

from ULM development.

REFERENCES 1. Segars JH, Parrott EC, Nagel JD, Guo XC, Gao X, Birnbaum LS et al. Proceedings from the Third National Institutes of

Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary

and future recommendations. Hum Reprod Update. 2014, 20(3), 309-33.

2. Bulun SE. Uterine fibroids. N Engl J Med. 2013, 3;369(14), 1344-55.

3. Blake RE. Leiomyomata uteri hormonal and molecular determinants of growth. J Natl Med Assoc. 2007, 99(10),1170-84.

4. Pakiz M, Potocnik U, But I. Solitary and multiple uterine leiomyomas among Caucasian women: two different disorders?

Fertil Steril. 2010, 94(6), 2291-5.

5. Walker CL, Stewart EA. Uterine fibroids: the elephant in the room. Science. 2005, 308, 1589–92.


42 | P a g e

Corresponding author: Matjaž Deželak, [email protected]

Abstract

A POLYMORPHISM LOCATED IN 3’UTR OF PITHD1 NEAR CANNABINOID RECEPTOR 2 GENE CNR2 IS

ASSOCIATED WITH SEVERE FORMS OF CROHN’S DISEASE AND CNR1 GENE EXPRESSION

Matjaž Deželak1, Žan Hribar1, Carina E. P. Kozmus1,2, Uroš Potočnik1,3

1Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Slovenia 2Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal 3Laboratory for Biochemistry Molecular Biology and Genomics, Faculty for Chemistry and Chemical Engineering, University of

Maribor, Maribor, Slovenia

ABSTRACT

Inflammatory bowel disease (IBD) is a chronic inflammatory and autoimmune disease of gastrointestinal tract. Crohn's disease (CD) and ulcerative colitis (UC) are the principal types of inflammatory bowel disease. CD patients that do not respond to standard therapy (corticosteroids, aminosalicylates, immunosuppressives, etc.) need treatment with biological drugs. The so-called ‘refractory CD patients’ can be regarded as a yet another subtype of IBD. The search for molecular biomarkers that would identify individuals refractory to standard therapy is mandatory to save costs on therapeutics as well as to improve patient’s well-being. Several reports have suggested that the endocannabinoid system (ECS) is involved in pathology of intestinal inflammation1 and that mRNA expression of cannabinoid receptor 2 gene CNR2 correlates with the seriousness of symptoms2. A single nucleotide polymorphism (SNP) rs4237 located in the 3’ untranslated region of PITHD1 and in the vicinity of CNR2, was reported to be an expression quantitative trait locus (eQTL) of CNR2 mRNA expression levels3. Therefore, we tested the hypothesis that rs4237 and CNR2 mRNA expression are specifically associated with refractory CD but not with other types of IBD and that rs4237 is associated with CNR2 mRNA expression. We studied case-control cohorts of 276 healthy individuals, 61 UC and 113 CD patients on standard therapy and 119 refractory CD patients on adalimumab (Humira®, Abbot Laboratories) therapy. Relative mRNA expressions of CNR1 and CNR2 were measured in PBMCs with qPCR using -ΔΔCq method4. Genotyping was performed by High Resolution Melting (HRM) analysis.

Between-cohort analyses confirmed that rs4237 was associated only with refractory CD (p = 3.50 10-2; odds ratio = 0.435; 95% confidence interval: 0.204-0.924) but not with other types of IBD. According to the recessive genetic model, individuals with CT or TT genotypes were more likely to be refractory to standard therapy (28.9%) compared to individuals with CC genotype (15.0%). CNR1 and CNR2 mRNA expressions

differed significantly between healthy individuals and refractory CD patients (p = 1.66 10-9 and p = 5.37 10-21, respectively). In refractory patients, median CNR1 and CNR2 mRNA expressions were lower (-1.236 ± 2.006 and -1.586 ± 2.071, respectively) compared to healthy individuals. Within-cohort analyses confirmed

eQTL relation between rs4237 and CNR1 mRNA expression (p = 2.10 10-2) specifically for refractory CD but not for other types of IBD. According to the recessive genetic model, refractory CD patients with CT or TT genotypes had lower median CNR1 mRNA expression (-1.510 ± 1.795) compared to patients with CC genotype (-0.266 ± 4.064). Our results suggest that CT or TT genotype of rs4237 in PITHD1 as well as 2- and 3-fold lower CNR1 and CNR2 mRNA expressions, respectively, increase the risk for refractory CD. In addition, eQTL relation between rs4237 and CNR1 mRNA expression was established for refractory CD patients.

REFERENCES 1. Fichna J, Bawa M, Thakur GA, et al. Cannabinoids alleviate experimentally induced intestinal inflammation by acting at central

and peripheral receptors. PLoS ONE 2007, 9(10), e109115.

2. Wright KL, Duncan M, Sharkey KA. Cannabinoid CB2 receptors in the gastrointestinal tract: a regulatory system in states of

inflammation. Br. J. Pharmacol. 2008, 153, 263-270.

3. Dixon AL, Liang L, Moffatt MF et al. A genome-wide association study of global gene expression. Nat. Genet. 2007, 39, 1202-7.

4. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))

method. Methods 2001, 25, 402-408.


43 | P a g e

ABSTRACTS and PAPERS of POSTERS

POPULATION GENETICS & GENOMICS

Taja Jeseničnik, Nataša Štajner, Sebastjan Radišek, Branka Javornik, Jernej Jakše

Discovery of small RNAs in Verticilliun nonalfalfae

Matej Babič, Tanja Kunej, Borka Jerman-Blažič

New method for statistical pattern recognition usage in cancer associated microRNAs

Blaž Skrlj, Nina Pirih, Tanja Kunej

Identification of non-synonymous polymorphisms within regions corresponding to

protein interaction sites

Marko Flajšman, Nataša Štajner, Igor Šantavec, Branka Javornik, Darja Kocjan Ačko

Genotypization and morphological characterization of six Slovenian landraces of proso

millet (Panicum miliaceum L.)


44 | P a g e

Corresponding author: Taja Jeseničnik, [email protected]

Abstract

DISCOVERY OF SMALL RNAs IN Verticilliun nonalfalfae

Taja Jeseničnik1, Nataša Štajner1, Sebastjan Radišek2, Branka Javornik1, Jernej Jakše1

1 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia 2 Plant Protection Department, Slovenian Institute for Hop Research and Brewing, Cesta Žalskega tabora 2, SI-3310, Žalec,

Slovenia

ABSTRACT

RNA interference is an evolutionary conserved eukaryotic mechanism regulating gene expression post-

transcriptionally1. The process of gene regulation is mediated by small RNAs2, including microRNA-like

small RNAs (milRNAs), recently shown to exist in filamentous fungi3,4,5,6. However, to date no milRNAs

have been reported in the phyto-pathogenic fungus Verticillium nonalfalfae, a soil borne plant

pathogen causing vascular wilt in many important crops worldwide. Two pathotypes of V. nonalfalfae,

mild strain and lethal strain, have been isolated from Slovenian hop fields, with the lethal strain causing

severe symptoms in hop plants, resulting in complete dieback of the plant7. With the accessible

genome sequence and detailed transcriptome of the two strains, we aim to identify milRNAs in of V.

nonalfalfae and to elucidate their possible involvement in the pathogenesis process. Discoveries of

novel microRNAs correlate with the advent of new NGS sequencing and bioinformatics technologies,

also applied for the purposes of this study. Two Slovenian isolates, Rec (mild) and T2 (lethal), were

acquired from the fungal genebank of the Slovenian Institute for Hop Research and Brewing. Small

RNA fractions were isolated from four different sources: spores, mycelia, mycelia grown on simulated

xylem fluid medium (SXM) and resting mycelia and used for small RNA library construction and

sequenced using the Ion Proton sequencing platform. Fungal milRNA precursors were predicted using

MIReNA software8 and the results were further validated and candidates selected manually using

criteria for plant miRNA candidates. Several candidate milRNA precursors were selected for both

pathotypes and have now been validated with stem-loop RT-PCR.

REFERENCES 1. Hannon GJ. RNA interference. Nature 2002, 418, 6894, 244-51.

2. Hamilton AJ, Blaucombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science

1999, 286, 5441, 950-2.

3. Zhou J, Fu Y, Xie J, Li B, Jiang D, Li G, Cheng J. Identification of microRNA-like RNAs in a plant pathogenic fungus

Sclerotinia sclerotiorum by high-throughput sequencing. Mol Genet Genomics 2012, 287, 4, 275-82.

4. Chen R, Jiang N, Jiang Q, Sun X, Wang Y, Zhang H, Hu Z. Exploring microRNA-like small RNAs in the filamentous fungus

Fusarium oxysporum. PLoS One 2014, 9, 8, e104956.

5. Mueth NA, Ramachandran SR, Hulbert SH. Small RNAs from the wheat stripe rust fungus (Puccinia striiformis f.sp. tritici).

BMC Genomics 2015, 16, 718.

6. Liu T, Hu J, Zuo Y, Jin Y, Hou J. Identification of microRNA-like RNAs from Curvularia lunata associated with maize leaf

spot by bioinformation analysis and deep sequencing. Mol Genet Genomics 2016, 291, 2, 587-96.

7. Radišek S, Jakše J, Simončič A, Javornik B.Characterization of Verticillium albo-atrum Field Isolates Using Pathogenicity

Data and AFLP Analysis. Plant Disease 2003, 87, 6, 633-638.

8. Mathelier A ,Carbone A. MIReNA: finding microRNAs with high accuracy and no learning at genome scale and from deep

sequencing data. Bioinformatics 2010, 26, 18, 2226-34.


45 | P a g e

Corresponding author: Matej Babič, [email protected]

Abstract

NEW METHOD FOR STATISTICAL PATTERN RECOGNITION USAGE IN CANCER ASSOCIATED

microRNAs

Matej Babič1, Tanja Kunej2, Borka Jerman-Blažič1

1Jozef Stefan Institute, Laboratory for Open Systems and Networks, Slovenia 2University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Slovenia

ABSTRACT

MicroRNAs (miRNAs) are small 22-25 nucleotides long noncoding RNA molecules that regulate gene

expression and that are present and stable in the bloodstream. They are evolutionally conserved, and

control gene expression in metazoan animals, plants, viruses, and bacteria primarily at post-

transcriptional and transcriptional levels. In this study, we present a novel 2D graphical representation

of miRNAs sequences based on graph theory. Graph theory is one of most tools in mathematics, which

are used in many cases, because we can simple present very complex problems. In recent years, a very

large variety of statistical methodologies, at various levels of complexity, have been put forward to

analyse genotype data and detect genetic variations that may be responsible for increasing the

susceptibility to cancer. In this article, we present new method for statistical pattern recognition.

Statistical pattern recognition relates to the use of statistical techniques for analysing data

measurements in order to extract information and make justified decisions. We considered

construction of n × 4 matrix to represent DNA primary sequences. Four column of matrix present 4

nucleotides of DNA base. First column present A, second C, third G and last column present T. We

traveled through DNA sequences to determinate each nucleotide from DNA base in matrix. Denoted

nucleotides present vertex of new graph. We calculated topological properties of this graph and

compared it for different genomic miRNAs sequences. The novelty of graphical representations of

miRNAs sequences is very useful for prediction cancer association. We tested the proposed method

on hsa-mir-423, hsa-mir-27a. The importance of topological modeling of miRNAs is distinguished, as

it presents simplified visual characteristics for the rapid and efficient modeling, comparison of miRNAs

and researching effort to establish their roles in cancer.

REFERENCES

1. Gregory, SG; Barlow, KF; McLay, KE; Kaul, R; Swarbreck, D; Dunham, A; Scott, CE; Howe, KL; et al. (2006). "The DNA

sequence and biological annotation of human chromosome 1". Nature. 441 (7091): 315–21. Bibcode:2006Natur.441..315G.

doi:10.1038/nature04727. PMID 16710414.


46 | P a g e

Corresponding author: Tanja Kunej, PhD, [email protected]

Article

IDENTIFICATION OF NON-SYNONYMOUS POLYMORPHISMS WITHIN REGIONS

CORRESPONDING TO PROTEIN INTERACTION SITES

Blaz Skrlj1, Nina Pirih1, Tanja Kunej1

1Department of Animal Science, Biotechnical Faculty, University of Ljubljana

Background: Protein-protein interactions (PPI) play an important role in function of all organisms and enable

understanding underlying metabolic processes. Predictions of protein interactions are an important aspect in proteomics,

as experimental methods may result in high degree of false positive results and are more expensive than computational

predictions. Prediction of PPIs has been a topic of increased interest in the last few years. Although there are many

databases collecting predicted PPIs, exploration of genetics information underlying PPI interactions has not been

investigated thoroughly. The aim of the present study was to identify: 1. genomic locations corresponding to regions

involved in predicted interaction sites, 2. Non-synonymous polymorphisms within interacting sites (PPI-SNPs), 3. examples

in which predicted PPIs sites overlap with previously reported experimentally validated interactions. Methods: Datasets

representing predicted PPIs were obtained from PiSITE database (http://pisite.hgc.jp). Non-synonymous polymorphisms

mapped on protein structural data (PDBs) were obtained from the UCSC’s ftp server. Polymorphism locations on protein

structures were mapped to predicted PPI regions in order to identify PPI-SNPs. Locations of experimentally validated PPIs

were extracted from published literature (http://www.ncbi.nlm.nih.gov/pubmed). Location of polymorphisms on 3D

protein structures was visualized using PyMol software. Results: We developed a scalable method for identification of PPI-

SNPs, which was further used to identify 662 polymorphisms located within the predicted PPI sites that map to 196 genes

that are associated with different diseases, mainly with different types of cancer. Additionally, 13 polymorphisms in seven

genes within predicted PPIs are also located within experimentally validated interaction regions. Conclusions: The present

study presents novel SNP prioritization approach for functional studies and potential candidates for disease biomarkers. INTRODUCTION

The study of protein interactions (PIs) provides insights into biological processes. Proteins can interact

with DNA, smaller ligands, RNA or with other proteins (protein-protein interactions; PPIs). Protein

interaction malfunctions can result in loss of phenotypic trait, which many times results in a disease1.

Relevance of protein interaction prediction is situated on the fact, that obtaining empirical evidence in

great quantity is currently a challenging task. Additionally, accuracy of high-throughput experiments

still yields many false-positive and false-negative results2. Using the data of predicted protein

interactions many a times provides additional evidence in explaining various biological phenomena3,

or facilitate planning experimental validation of experimental PPIs4. Use of modern prediction

techniques such as machine learning, graph theory, Bayesian prediction and pattern recognition have

been used in tools, such as Struct2Net5, PiPs6, HitPredict7, PRISM8, DOMINE9, ProBis10, and PiSITE11.

Previous studies on domain altering SNPs in human proteome12 identified many polymorphisms which

alter specific protein domains and can therefore have clinically significant effects. Non-synonymous

polymorphisms (nsSNP) have been shown to cause pathological effects, for example in

hypercholesterolemia13, incident coronary heart disease14, breast cancer15 and many other types of

cancer.

Prediction of the effect of amino acid substitution on protein function is an important task in systems

biology. One of the most widely used tools in this field is SIFT algorithm, which uses homology based

approach to identify amino acid loci, which could potentially affect protein function. SIFT tool has

already been successfully used to analyze effects of possible cancer mutations16; yet identified

mutations were not directly associated with protein-protein interaction sites. However, significant 3D

clustering of missense mutations in several previously known oncoproteins has been detected17. In our

previous study we reanalyzed genomic regions involved in interactions between transcription factor

and its targets and identified polymorphisms located within hypoxia response elements (HREs)18.

http://pisite.hgc.jp/


47 | P a g e

Although there are already many datasets providing predicted PPI interactions, those predictions

remain in the protein domain – they are not associated with underlying nucleotide sequence and

genetic variability. To our knowledge, there are no reported studies related with polymorphisms

located within regions corresponding to predicted protein-protein interaction information performed

for nsSNPs, mapped onto protein structure (PDB)16.

Protein-protein interaction data used in this study was obtained from the PiSITE database. Interactions

were predicted using BLAST queries, residue specific interactions were identified based on distance

criteria, where distance between residues needed to be smaller than 4 Å11. The aim of this study was

to identify nsSNPs, occurring within predicted protein-protein interaction sites.

METHODS

Non-synonymous polymorphisms mapped onto PDB structures (n=11551) were obtained from UCSC

institute’s ftp server19. Regions involved in predicted protein-protein interactions were obtained from

PiSITE11. Mutations’ datasets were preprocessed and further merged using algorithms, implemented

in R language (https://www.r-project.org/). Using Python scripting (https://www.python.org/), we

extracted all interactions from the PiSITE dataset, consisting of 110325 chains. Association of genes

with diseases was analyzed using functional annotation tool DAVID

(https://david.ncifcrf.gov/home.jsp). The predicted protein-protein interaction interfaces were further

analyzed for overlap with experimentally validated interactions in previously published literature

(http://www.ncbi.nlm.nih.gov/pubmed). In addition, we selected nsSNPs that are located within

predicted as well as experimentally validated PPI regions. Selected results were visualized using open-

source PyMol software (https://sourceforge.net/projects/pymol/).

RESULTS

In the present study we identified nsSNPs located within regions corresponding to predicted protein-

protein interaction interfaces. For some selected cancer genes we also confirmed that predicted

interaction sites overlap with previously reported experimentally validated interaction sites. Main

workflow of the process is presented in Figure 1.

Figure 1: Main workflow of the study.

Mapping of nsSNPs to predicted interaction sites resulted in the catalog of 662 nsSNPs with matching

reference SNP ID (rs) accession numbers from dbSNP. Collected 662 PPI-SNPs within predicted

interaction sites map to 196 protein-coding genes. Further data processing revealed that from 196

identified genes, 48 are statistically significant associated with at least one type of cancer. Additionally,

genes were also associated with Alzheimer’s disease, rheumatoid arthritis, diabetes type 1 and type 2,

asthma, cardiovascular disease, cholesterol, depression, and Crohn’s disease. Ten cancer associated

genes with the highest number of SNPs located within predicted PPIs are presented in Table 1.

Table 1: Ten genes with the highest number of SNPs, located within predicted PPIs..

https://www.r-project.org/

https://www.python.org/

https://david.ncifcrf.gov/home.jsp

http://www.ncbi.nlm.nih.gov/pubmed

https://sourceforge.net/projects/pymol/


48 | P a g e

Gene name CFTR TCAP XRCC5 FGFR2 MICA SERPINA1 VHL TP53 BRCA1

Number of PPI-SNPs

5 5 5 6 6 8 8 10 15

rs SNP ID numbers

(examples)

rs74571530

rs35032490

rs1800100

rs80055610

rs11531593

rs45495192

rs45458802

rs17851031

rs45614536

rs45513698

rs1805380

rs11558396

rs2287558

rs11575909

rs61758380

rs55977237

rs755793

rs77543610

rs4647921

rs79184941

rs55689343

rs3819268

rs17200179

rs17206575

rs17206589

rs17200207

rs17200186

rs17580

rs28929471

rs28929473

rs11575873

rs1131154

rs55819880

rs72552401

rs12077

rs17855706

rs5030812

rs5030818

rs28940297

rs5030811

rs5030804

rs5030805

rs5030822

rs28934875

rs28934572

rs28934575

rs11540652

rs28934573

rs28934576

rs28934578

rs28934571

etc.

rs80357316

rs80357031

rs80357054

rs80357017

rs80357406

rs80357438

rs1800062

rs80356929

etc.

Legend: CFTR: Cystic fibrosis transmembrane conductance regulator; TCAP: Telethonin, XRCC5: X-ray repair

cross-complementing protein 5, FGFR2: Fibroblast growth factor receptor 2, MICA: MHC class I polypeptide-

related sequence A, SERPINA1: Alpha-1-antitrypsin, VHL: von Hippel-Lindau tumor suppressor; TP53: tumor

protein p53; BRCA1: BRCA1, DNA repair associated.

Using data from the literature we found 7 genes in which predicted PPI sites also corresponded to

experimentally validated interaction sites: TCAP, APOA1, CRP, ESR1, TP53, VHL, and SHFM1. Out of 662

PPI-SNPs 13 nsSNPs are present within predicted and validated PPIs (Table 2).

Table 2: Identified nsSNPs in genes associated with cancer that are located within predicted as well as experimentally

validated protein-protein interaction interfaces.

Gene name rs SNP ID numbers

Associated cancer type (According to DAVID functional annotation analysis).

Interaction sites found in the literature

TCAP rs45495192 rs45614536 rs45513698

breast cancer 1–53 (binding to the very N-terminus of titin20)

APOA1 rs28931573 colorectal cancer 161–240 (binding to NDRG121)

CRP rs34200896 lung cancer 109-132 (constitutive binding region22)

ESR1 rs79374934

bone cancer, breast cancer, colon cancer, endometrial cancer, prostate cancer, uterine cancer etc.

185–595 (binding to MUC1-CD23) 410–731 (binding to HPIP24)

TP53 rs28934576

bladder cancer, leukemia, lung cancer, brain cancer, breast cancer, cervical cancer, stomach cancer etc.

256 – 294 (Binding to p120E4F25)

VHL rs17855706 rs5030812 rs5030818 rs28940297

brain cancer, breast cancer, renal cancer, kidney cancer

100-155 (binding to TRiC26) 157–172 (binding to elongin BC26),

SHFM1 rs11553943 rs1802882

breast cancer 7-25, 37-63 (binding to BRCA227)

For example, in VHL gene there are four nsSNPs within predicted PPI and also located within previously

experimentally validated interaction regions. Graphic presentation for VHL gene is presented in Figure

2. Polymorphisms are located on locus 110 (rs17855706), 115 (rs5030812), 161 (rs5030818) and 163

(rs28940297). Experimentally validated PPI sites encompass amino acids 100-155 and 157-17226.


49 | P a g e

Figure 2: Visualization of polymorphisms within predicted and experimental interaction regions in VHL gene (PDB accession

1lm8, chain V) on modified figure from PiSITE server. Predicted PPI region is marked with green color and

experimental PPI region with blue line. Polymorphisms are marked with red boxes.

Three dimensional visualization of VHL gene and four polymorphisms located within predicted and

validated interaction sites are presented in Figure 3.

Figure 3: Representation of different layers of interaction information in this study on example protein VHL. Four

polymorphisms located within both, predicted and experimentally validated interaction sites are marked in pink

color: rs17855706 (His110Tyr), rs5030812 (His115Arg), rs5030818 (Arg161Ter) and rs28940297 (Leu163Pro).

DISCUSSION

Identification of nsSNPs located within predicted protein-protein interaction sites provides additional

information on biological phenomena and enables more detailed explanation of disease development.

Within locations of predicted protein interactions we identified 662 polymorphisms mapping to 196

genes, which showed significant clustering in terms of cancer and other disease associations. The


50 | P a g e

results of the present study are in concordance with an observation by Kamburov et al. (2015), who

proved significant clustering patterns of polymorphisms emerged in cancer related protein

interactions. Oncoproteins VHL, TP53 and BRCA1 were in the present study additionally confirmed as

proteins with the highest number of predicted PPI-SNPs. In addition, regions involved in predicted PPI

also overlapped with experimentally validated interactions in seven proteins, including VHL and TP53.

By literature mining we found two interaction sites for protein VHL. VHL binds to elongin BC via a linear

sequence known as “BC box”, consist of amino acids 157–17226. We have detected two nsSNPs

(rs5030818, rs28940297) located within this region, which is known as a hot spot for disease-causing

inherited point mutations26. Furthermore we have identified two nsSNPs (rs17855706, rs5030812) in

the region of VHL comprising amino acids 100-155, that mediates TRiC binding26. Within previously

reported experimentally validated interaction region of oncoprotein TP53, we have identified one

nsSNP (rs28934576). This TP53 region is important for binding to the E1A-regulated transcription factor

p120E4F, a transcriptional repressor of the adenovirus E4 promoter. The interaction involves carboxy-

terminal half of p120E4F and sequences located at the end of the sequence-specific DNA-binding

domain of TP53, which encompassing amino acids 256 – 29425.

Similarly to our previous SNP prioritization studies in which we collected polymorphisms located within

miRNA genes28 and within transcription factor-target interaction18 in the present study we collected

polymorphisms, located within predicted PPI sites, associated with cancer risk. Additionally we have

also confirmed that predicted interaction sites overlap with experimentally validated interactions in

previously published literature. In conclusion, we identified 13 nsSNPs located within both, predicted

and previously experimentally validated interaction sites that map to seven cancer associated genes.

In addition, an investigation whether other identified nsSNPs within PPI sites are associated with

cancer should also follow. The results of the present preliminary study could serve as a basis for

planning more targeted experimental designs, functional experiments and might present novel drug

targets and therapeutic strategies for cancer.

REFERENCES

1. Gonzalez M W, Kann M. G. Chapter 4: Protein interactions and disease. PLoS Comput Biol. 2012, 8, 12.

2. Rao V S, Srinivas K, Sujini G N, Kumar G N. Protein-protein interaction detection: methods and analysis. Int J Proteomics.

2014.

3. Zahiri J, Bozorgmehr J H, Masoudi-Nejad A. Computational prediction of protein-protein interaction networks: algo-

rithms and resources. Curr Genomics. 2013, 14, 6, 397-414.

4. Jessulat M, Pitre S, Gui Y, Hooshyar M, Omidi K, Samanfar B et al. Recent advances in protein-protein interaction

prediction: experimental and computational methods. Expert opinion on drug discovery. 2011, 6, 9, 921-935.

5. Singh R, Park D, Xu J, Hosur R, Berger B. Struct2Net: a web service to predict protein-protein interactions using a

structure-based approach. Nucleic Acids Res. 2010, 38, 508-515.

6. McDowall M D, Scott M S, Barton G J. PIPs: human protein-protein interaction prediction database. Nucleic Acids Res.

2009, 37, 651-656.

7. López Y, Nakai K, Patil A. HitPredict version 4: comprehensive reliability scoring of physical protein-protein interactions

from more than 100 species. Database (Oxford). 2015.

8. Baspinar A, Cukuroglu E, Nussinov R, Keskin O, Gursoy A. PRISM: a web server and repository for prediction of protein-

protein interactions and modeling their 3D complexes. Nucleic Acids Res. 2014, 42, 285-289.

9. Yellaboina S, Tasneem A, Zaykin D V, Raghavachari B, Jothi R. DOMINE: a comprehensive collection of known and

predicted domain-domain interactions. Nucleic Acids Res. 2011, 39, 730-735.

10. Konc J, Janežič D. ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites. Nucleic

Acids Res. 2014, 42, 215-220.

11. Higurashi M, Ishida T, Kinoshita K. PiSite: a database of protein interaction sites using multiple binding states in the

PDB." Nucleic acids research. 2009, 37, 360-364.

12. Liu Y, Tozeren A. Domain altering SNPs in the human proteome and their impact on signaling pathways. PLoS One. 2010,

5, 9.


51 | P a g e

13. Angarica V E, Orozco M, Sancho J. Exploring the complete mutational space of the LDL receptor LA5 domain using

molecular dynamics: linking SNPs with disease phenotypes in familial hypercholesterolemia. Hum Mol Genet. 2016, 25,

6, 1233-1246.

14. Jensen M K, Pers T H, Dworzynski P, Girman C J, Brunak S, Rimm E B. Protein interaction-based genome-wide analysis of

incident coronary heart disease. Circ Cardiovasc Genet. 2011, 4, 5, 549-556.

15. Milne R L, Burwinkel B, Michailidou K, Arias-Perez J I, Zamora M P, Menéndez-Rodríguez P et al. Common non-

synonymous SNPs associated with breast cancer susceptibility: findings from the Breast Cancer Association Consortium.

Hum Mol Genet. 2014, 23, 22, 6096-6111.

16. Kumar P, Henikoff S, Ng P C. Predicting the effects of coding non-synonymous variants on protein function using the

SIFT algorithm. Nature protocols. 2009, 4, 7, 1073-1081.

17. Kamburov A, Lawrence M S, Polak P, Leshchiner I, Lage K, Golub T R et al. Comprehensive assessment of cancer

missense mutation clustering in protein structures. Proc Natl Acad Sci U S A. 2015, 112, 40, 5486-5495.

18. Slemc L, Kunej T. Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response

element (HRE) sites and initiative for standardization of reporting in scientific literature. Tumor Biology. In press.

19. Ryan M, Diekhans M, Lien S, Liu Y, Karchin R. LS-SNP/PDB: annotated non-synonymous SNPs mapped to Protein Data

Bank structures. Bioinformatics. 2009, 25, 11, 1431-1432.

20. Pinotsis N, Petoukhov M, Lange S, Svergun D, Zou P, Gautel M et al. Evidence for a dimeric assembly of two

titin/telethonin complexes induced by the telethonin C-terminus. J Struct Biol. 2006, 155, 2, 239-250.

21. Hunter M., Angelicheva D, Tournev I, Ingley E, Chan D C, Watts G F et al. NDRG1 interacts with APO A-I and A-II and is a

functional candidate for the HDL-C QTL on 8q24. Biochem Biophys Res Commun. 2005, 332, 4, 982-992.

22. Zhang J, Yang L, Ang Z, Yoong S L, Tran T T, Anand G S et al. Secreted M-ficolin anchors onto monocyte transmembrane

G protein-coupled receptor 43 and cross talks with plasma C-reactive protein to mediate immune signaling and regulate

host defense. J Immunol. 2010, 185, 11, 6899-6910.

23. Wei X, Xu H, Kufe D. MUC1 oncoprotein stabilizes and activates estrogen receptor alpha. Mol Cell. 2006, 21, 2, 295-305.

24. Manavathi B, Acconcia F, Rayala S K, Kumar R. An inherent role of microtubule network in the action of nuclear

receptor. Proc Natl Acad Sci U S A. 2006, 103, 43, 15981-15986.

25. Sandy P, Gostissa M, Fogal V, Cecco L D, Szalay K, Rooney R J et al. p53 is involved in the p120E4F-mediated growth

arrest. Oncogene. 2000, 19, 2, 188-199.

26. Feldman D E, Thulasiraman V, Ferreyra R G, Frydman J. Formation of the VHL-elongin BC tumor suppressor complex is

mediated by the chaperonin TRiC. Mol Cell. 1999, 4, 6, 1051-1061.

27. Yang, H, Jeffrey P D, Miller J, Kinnucan E, Sun Y, Thoma, N H et al. BRCA2 function in DNA binding and recombination

from a BRCA2-DSS1-ssDNA structure. Science. 2002, 297, 5588, 1837-1848.

28. Zorc M, Obsteter J, Dovc P, Kunej T. Genetic Variability of MicroRNA Genes in 15 Animal Species. J Genomics. 2015, 3,

51-56.


52 | P a g e

Corresponding author: Darja Kocjan Ačko, [email protected]

Abstract

GENOTYPIZATION AND MORPHOLOGICAL CHARACTERIZATION OF SIX SLOVENIAN

LANDRACES OF PROSO MILLET (Panicum miliaceum L.)

Marko Flajšman1, Nataša Štajner1, Igor Šantavec1, Branka Javornik1, Darja Kocjan Ačko1 1University of Ljubljana, Biotechnical Faculty, Agronomy Department, Jamnikarjeva 101, Ljubljana SI-1000, Slovenia

ABSTRACT

Proso millet (Panicum miliaceum L.) is one of the world's oldest cultivated cereals. It appeared as a staple crop in northern China 10,000 years ago1 and later spread to other parts of the world, including Slovene territory where it was grown as early as a thousand years B.C. by the Celts2. Proso millet has two main advantages over other cereals: firstly, it is considered a health-food crop owing to its unique nutritional value3 and secondly, it is very tolerant of various abiotic stresses, e.g., salinity and drought. In addition, it can grow well in poor soils4. In Slovenia, the cultivation of millet reached its peak in the 19th century, covering 18,000 ha. In 2016, proso millet is cultivated on only around 200 ha, mostly by organic farmers, who use landraces of yellow and white proso millet from Prekmurje and Gorenjska regions, the Austrian cultivar 'Kornberger Mittelfrühe' domesticated in Slovenia as 'Kornberg millet' and, in recent years, also the cultivar 'Sonček'2. In the current study, we analysed the genetic diversity of six Slovenian landraces of proso millet together with the cultivar 'Sonček' by utilizing both molecular markers and morphological traits. The use of microsatellites (SSRs), which have proved to be a powerful tool for genetic diversity analyses in various crops, also resulted in high discrimination with proso millet. We successfully amplified 11 SSR loci and, of these, 7 loci were polymorphic. Only accessions of the cultivar 'Sonček' formed a uniform cluster, all other landraces were very diverse. Nevertheless, we were able to differentiate six different clusters of genotypes, representing six landraces, which differ also in the morphology of the panicle. Moreover, yield stability and 1000-seed weight, as well as other morphological characteristics, were determined in a 2-year field experiment, also showing very diverse results between years as well as among samples within some landraces. 1. Lu H, Zhang J, Liu K-B, Wu N, Li Y, Zhou K, Ye M, Zhang T, Zhang H, Yang X. Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proc Natl Acad Sci USA. 2009, 106, 7367–7372. 2. Kocjan Ačko D. Importance and possibilities of proso millet (Panicum miliaceum L.) production for human nutrition and animal feed in Slovenia. Int. j. food, agric. environment. 2012, 10(2), 636-640. 3. Liu M, Qiao Z, Zhang S, Wang Y, Lu P. Response of broomcorn millet (Panicum miliaceumL.) genotypes from semiarid regions of China to salt stress. Crop J. 2015, 3, 57–66. 4. Sabir P, Ashraf M, Akram NA. Accession variation for salt tolerance in proso millet (Panicum miliaceum L.) using leaf proline content and activities of some key antioxidant enzymes. J. Agron. Crop Sci. 2011, 197, 340–347.


53 | P a g e

SPONSORS

slovenian society of human genetics

Documents